Biologically active spermidine analogues, pharmaceutical compositions and methods of treatment

ABSTRACT

Polyamines having the formula:                    
     or a salt thereof with a pharmaceutically acceptable acid wherein: R 1 -R 5  may be the same or different and are alkyl, aryl, aryl alkyl, cycloalkyl or hydrogen; at least one of R 1  and R 2  and at least one of R 4  and R 5  are not hydrogen, and any of the alkyl chains may optionally be interrupted by at least one etheric oxygen atom, excluding N 1 ,N 3 -diethylspermidine and N 1 ,N 3 -dipropylspermidine; and 
     A and B are bridging groups which effectively maintain the distance between the nitrogen atoms such that the polyamine: (i) is capable of uptake by a target cell upon administration of the polyamine to a human or non-human animal; and (ii) upon uptake by the target cell, competitively binds via an electrostatic interaction between the positively charged nitrogen atoms to substantially the same biological counter-anions as the intracellular natural polyamines in the target cell.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel polyamines useful as activeingredients in pharmaceutical compositions and therapeutic methods oftreatment.

2. Description of the Prior Art

Because of the sustained increases in polyamine biosynthesis inpre-neoplastic and neoplastic tissues, a great deal of attention hasbeen directed to the polyamine biosynthetic network as a target inanti-neoplastic strategies [Pegg, “Polyamine Metabolism and ItsImportance in Neoplastic Growth and as a Target for Chemotherapy,”Cancer Res., Vol. 48, pages 759-774 (1988); and Marton et al,“Directions for Polyamine Research,” J. Cell Biochem., Vol. 45, pages7-8 (1991)]. Initial work focused on the design and synthesis ofcompounds which would inhibit L-ornithine decarboxylase (ODC) [Bey etal, “Inhibition of Basic Amino Acid Decarboxylases Involved in PolyamineBiosynthesis,” Inhibition of Metabolism Biological Significance andBasis for New Therapies, McCann et al, eds.; Academic Press: Orlando,Fla., pages 1-32 (1987)] and S-adenosyl-L-methionine decarboxylase(AdoMetDC) [Pegg, Cancer Res., Vol. 48, supra; and Williams-Ashman etal, “Methylgly-oxal Bis(guanylhydrazone) as a Potent Inhibitor ofMammalian and Yeast S-Adenosylmethionine Decarboxylases,” Biochem.Biophys. Res. Commun., Vol. 46, pages 288-295 (1972)]. Some success wasachieved through this approach in that difluoromethylornithine (DFMO),an ODC inhibitor, and methylglyoxylbis(guanylhydrazone) (MGBG), anAdoMetDC inhibitor, were effective against both in vivo and in vitrotumors [Sunkara et al, “Inhibitors of Polyamine Biosynthesis: Cellularand In Vivo Effects on Tumor Proliferation,” Inhibition of PolyamineMetabolism Biological Significant Cause and Basis for New Therapies,McCann et al, eds.; Academic Press: Orlando, Fla., pages 121-140 (1987);and Pegg et al, “S-Adenosylmethionine Decarboxylase as an Enzyme Targetfor Therapy,” Pharmacol. Ther., Vol. 56, pages 359-377 (1992)]. However,clinical trials did not mirror the success realized in the modelsystems; the drug either was too toxic as with MGBG [Pegg et al,Biochem. Pharmacol., Vol. 27, pages 1625-1629 (1978)] or was unable toshow significant impact on tumors in humans as with DFMO [Schecter etal, “Clinical Aspects of Inhibition of Ornithine Decarboxylase withEmphasis on the Therapeutic Trials of Eflornithine (DFMO) in Cancer andProtozoan Diseases,” Inhibition of Polyamine Metabolism. BiologicalSignificance and Basis for New Therapies, McCann et al, eds.; AcademicPress: Orlando, Fla., pages 345-364 (1987)]. One of the problems withthe target enzymes ODC and AdoMetDC is associated [Seiler et al,“Polyamine Transport in Mammalian Cells,” Int. J. Biochem., Vol. 22,pages 211-218 (1990)] with their very short half-lives, i.e., about 20minutes. This can translate into a protracted exposure requirement forpatients which is a less than desirable situation. Nonetheless, bothDFMO and MGBG served well as proof of principle that the polyaminebiosynthetic network was an excellent target in the design ofanti-cancer drugs.

It would thus be desirable to design polyamine analogues which would beincorporated via the polyamine transport apparatus and, once in thecell, would find their way to the same subcellular distribution sites asthe normal polyamines do, but would be unable to be further processed[Jännë et al, “Polyamines in Rapid Growth and Cancer,” Biochim. Biophys.Acta, Vol. 473, page 241 (1978); and Porter et al, “Enzyme Regulation asan Approach to Interference with Polyamine Biosynthesis—an Alternativeto Enzyme Inhibition,” Enzyme Regul., Vol. 27, pages 57-79 (1988)]. Theywould appear enough like the natural polyamines to shut down polyamineenzymes just as when the cells are exposed to exogenous spermine.

Thus, a series of terminally N-alkylated tetraamines, which exhibitanti-neoplastic activity against a number of murine and human tumorlines both in vitro and in vivo, were assembled [Bergeron et al,“Synthetic polyamine analogues as antineoplastics,” J. Med. Chem., Vol.31, pages 1183-1190 (1988); Bergeron et al, “AntiproliferativeProperties of Polyamine Analogues: a Structure-Activity Study,” J. Med.Chem., Vol. 37, pages 3464-3476 (1994); Bernacki et al, “AntitumorActivity of N,N′-Bis(ethyl)spermine Homologues Against Human MALME-3Melanoma Xenografts,” Cancer Res., Vol. 52, pages 2424-2430 (1992);Porter et al, “Biological Properties of N⁴-Spermidine Derivatives andTheir Potential in Anti-cancer Chemotherapy,” Cancer Res., Vol. 42,pages 4072-4078 (1982); and Porter et al, “Biological Properties of N⁴-and N¹,N⁸-Spermidine Derivatives in Cultured L1210 Leukemia Cells,”Cancer Res., Vol. 45, pages 2050-2057 (1985)]. These tetraamines havebeen shown to utilize the polyamine transport apparatus forincorporation [Bergeron et al, J. Med. Chem., Vol. 37, supra; and Porteret al, “Aliphatic Chain Length Specific of the Polyamine TransportSystem in Ascites L1210 Leukemia Cells,” Cancer Res., Vol. 44, pages126-128 (1984)], deplete polyamine pools [Bergeron et al, “Role of theMethylene Backbone in the Antiproliferative Activity of PolyamineAnalogues on L1210 Cells,” Cancer Res., Vol. 49, pages 2959-2964(1989)], drastically reduce the level of ODC [Pegg et al, “Control ofOrnithine Decarboxylase Activity in α-Difluoromethylornithine-ResistantL1210 Cells by Polyamines and Synthetic Analogues,” J. Biol. Chem., Vol.263, pages 11008-11014 (1988); and Porter et al, “Relative Abilities ofBis(ethyl) Derivatives of Putrescine, Spermidine and Spermine toRegulate Polyamines Biosynthesis and Inhibit L1210 Leukemia CellGrowth,” Cancer Res., Vol. 47, pages 2821-2825 (1987)] and AdoMetDCactivities [Pegg et al, J. Biol. Chem., Vol. 263, supra; and Porter etal, Cancer Res., Vol. 47, supra] and in some cases to up-regulatespermidine/spermine/N¹-acetyltransferase (SSAT) [Pegg et al, “Effect ofN¹,N¹²-Bis(ethyl)spermine and Related Compounds on Growth and PolyamineAcetylation, Content and Excretion in Human Colon Tumor Cell,” J. Biol.Chem., Vol. 264, pages 11744-11749 (1989); Casero et al, “DifferentialInduction of Spermidine/Spermine N¹-Acetyltransferase in Human LungCancer Cells by the Bis(ethyl)polyamine Analogues,” Cancer Res., Vol.49, pages 3829-3833 (1989); Libby et al, “Major Increases inSpermidine/Spermine-N¹-Acetyltransferase by Spermine Analogues and TheirRelationship to Polyamine Depletion and Growth Inhibition in L1210Cells,” Cancer Res., Vol. 49, pages 6226-6231 (1989); Libby et al,“Structure-Function Correlations of Polyamine Analog-Induced Increasesin Spermidine/Spermine Acetyltransferases Activity,” Biochem.Pharmacol., Vol. 38, pages 1435-1442 (1989); Porter et al, “CorrelationsBetween Polyamine Analog-Induced Increases in Spermidine/SpermineN-Acetyltransferase Activity, Polyamine Pool Depletion and GrowthInhibition in Human Melanoma Cell Lines,” Cancer Res., Vol. 51, pages3715-3720 (1991); Fogel-Petrovic et al, “Polyamine and Polyamine AnalogRegulation of Spermidine/Spermine N¹-Acetyltransferase in MALME-3M HumanMelanoma Cells,” J. Biol. Chem., Vol. 268, pages 19118-19125 (1993); andShappell et al, “Regulation of Spermidine/Spermine N¹-Acetyltransferaseby Intra-cellular Polyamine Pools-Evidence for a Functional Role inPolyamine Homeostasis,” FEBS Lett., Vol. 321, pages 179-183 (1993)].Interestingly, on incorporation of the tetraamine analogues, the totalpicoequivalents of charge associated with the analogues, as well as thenatural polyamines, is maintained for about 24 hours. Thus, as the cellis incorporating n picoequivalents of drug, it is excreting npicoequivalents of natural polyamines.

Very small structural alterations in these spermine analogues andhomologues result in substantial differences in their biologicalactivity [Bergeron et al, Cancer Res., Vol. 49, supra]. For example,while the tetraamines N¹,N¹²-diethyl-spermine (DESPM),N¹,N¹¹-diethylnorspermine (DENSPM) and N¹,N¹⁴-diethylhomospermine(DEHSPM) suppress ODC and AdoMetDC to about the same level at equimolarconcentrations, the effect of both DESPM and DEHSPM on cell growthoccurs earlier than that observed for DENSPM. The K_(i) value of DENSPMis over 10 times as great [Bergeron et al, Cancer Res., Vol. 49, supra]as those of DESPM and DEHSPM for the polyamine transport system.However, the most notable difference between the three analogues isrelated to their ability to stimulate SSAT [Casero et al, Cancer Res.,Vol. 49, supra; Libby et al, Cancer Res., Vol. 49, supra; Libby et al,Biochem. Pharmacol., Vol. 38, supra; and Porter et al, Cancer Res., Vol.51, supra]. The tetraamine DENSPM up-regulates SSAT by 1200 fold inMALME-3 cells, while DESPM and DEHSPM stimulate SSAT by 250- and30-fold, respectively [Porter et al, Cancer Res., Vol. 51, supra]. Thus,the impact of the tetraamine compounds on cell growth was shown to bedependent on: the distance between the nitrogens; the nature of theterminal alkyl substituents [Bergeron et al, J. Med. Chem., Vol. 37,supra] and, most importantly, on the charge status of the molecules[Bergeron et al, “The Role of Charge in Polyamine Analogue Recognition,”J. Med. Chem., Vol. 38, pages 2278-2285 (1995)].

It was decided to establish whether or not a similar structure activityrelationship exists for triamines, i.e., analogues of spermidine. Theimportance of this issue is underscored by the tremendous difference intoxicity between the triamines and tetraamines in general. Triamines aremuch less toxic, thus making them of potentially useful therapeuticvalue [Bergeron et al, “Metabolism and Pharmacokinetics ofN¹,N¹¹-Diethylnorspermine,” Drug Metab. Dispos., Vol. 23, pages1117-1125 (1995)].

It is, therefore, an object of the present invention to provide certainnovel triamines possessing biological activity, in particular,anti-neoplastic activity.

SUMMARY OF THE INVENTION

This and other objects are realized by the present invention, oneembodiment of which relates to polyamines not occurring in nature havingthe formula:

or a salt thereof with a pharmaceutically acceptable acid wherein:

R₁-R₅ may be the same or different and are alkyl, aryl, aryl alkyl,cycloalkyl or hydrogen; at least one of R₁ and R₂ and at least one of R₄and R₅ are not hydrogen, and any of the alkyl chains may optionally beinterrupted by at least one etheric oxygen atom, excludingN¹,N³-diethylspermidine and N¹,N³-dipropylspermidine; and

A and B may be the same or different and are bridging groups includingunsubstituted heterocyclic bridging groups which effectively maintainthe distance between the nitrogen atoms such that the polyamine: (i) iscapable of uptake by a target cell upon administration of the polyamineto a human or non-human animal; and (ii) upon uptake by the target cell,competitively binds via an electrostatic interaction between thepositively charged nitrogen atoms to substantially the same biologicalcounter-anions as the intra-cellular natural polyamines in the targetcell, provided that where A or B is a heterocyclic bridging group, thebridging group is an unsubstituted heterocyclic group incorporating saidN¹, N² or N³ atoms in the heterocyclic ring as an unsubstituted N atom;the polyamine, upon binding to the biological counter-anion in the cell,functions in a manner biologically different than the intracellularpolyamines.

A further embodiment of the invention concerns a pharmaceuticalcomposition in unit dosage form comprising a pharmaceutically acceptablecarrier and a pharmaceutically effective amount of a polyamine asdescribed above or a salt thereof with a pharmaceutically acceptableacid.

An additional embodiment of the invention comprises a method of treatinga human or non-human patient in need thereof comprising administeringthereto a pharmaceutically effective amount of a polyamine describedabove or a salt thereof with a pharmaceutically acceptable acid.

Other embodiments of the invention will become apparent from thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 depict reaction schemes for the syntheses of the polyamines ofthe invention.

FIGS. 6(a) and 6(b) depict the structure-activity relationship betweentriamine analogues and tetraamine analogues, respectively, and SSATup-regulation.

FIG. 7 elaborates the metabolic transformation of the triamineanalogues.

FIG. 8 depicts the structure-activity relationship between the triamineanalogues and K_(i) values.

FIG. 9(a) represents the structure-activity relationship between thetriamine analogues and the IC₅₀ values.

FIG. 9(b) illustrates the structure-activity relationship between thetetraamine analogues and the IC₅₀ values.

DETAILED DESCRIPTION OF THE INVENTION

In the polyamines of the invention, as described in the above structuralformula, R₁-R₆ may be alkyl, e.g., methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl; aryl, e.g., phenyl, p-tolyl,2,4,6-trimethylphenyl; aryl alkyl, e.g., benzyl, α-phenethyl,β-phenethyl; cycloalkyl, e.g., cyclohexyl, cyclobutyl, cyclopentyl,cycloheptyl; any of the foregoing wherein the alkyl chain is interruptedby etheric oxygen, e.g., CH₃O(CH₂)₂—, CH₃O(CH₂)₂O(CH₂)₂—,CH₃O(CH₂)₂O(CH₂)₂O(CH₂)₂—; or hydrogen.

Except where R₁-R₆ are hydrogen or etheric substituents, each arehydrocarbyl and may have from about 1 to about 10 carbon atoms, it beingunderstood that the size of the substituents will be tailored in eachcase to ensure that the polyamine is capable of uptake by the targetcell and, upon uptake, will competitively bind with the intracellularcounter-anions as described above.

The bridging groups A and B may be the same or different and may bealkylene having 1-8 carbon atoms, e.g., methylene, trimethylene,tetramethylene, pentamethylene; branched alkylene, e.g.,—CH(CH₃)CH₂CH₂-, —CH₂CH(CH₃)CH₂—, —CH(CH₃)CH₂CH₂—, —CH₂CH(CH₃)CH₂CH₂—;arylalkylene, e.g., —CH(Ph)CH₂CH₂—, —CH₂CH(Ph)CH₂—, —CH(Ph)CH₂CH₂CH₂—,—CH₂CH(Ph)CH₂—CH₂—; cycloalkylene, e.g., cyclohexylene, cis- andtrans-1,3-cyclohexylene, 1,4-cyclohexylene, 1,3-cyclopentylene;heterocyclic groups which incorporate within the ring one of thenitrogen atoms of the polyamine [e.g.,

it being understood that the heterocyclic nitrogen group may be locatedat the terminal end(s) or within the interior of the polyamine.

Those skilled in the art will appreciate that it is only necessary thatthe bridging groups be selected so as to ensure uptake by the cell andcompetitive binding to the intracellular counter-anion as describedabove.

At physiological pH's, the naturally occurring polyamines and theanalogs of the present invention are largely in a protonated state[Bergeron et al, “Hexahydropyrimidines as masked spermidine vectors indrug delivery,” Bioorg. Chem., Vol. 14, pages 345-355 (1986)]. At acellular level, these polycations can bind to a collection of singleunconnected anions or to anions tethered to a single biomolecule, e.g.,the phosphates on a nucleic acid.

If there is any significance to the role of charge interaction in thebiological properties of the polyamine analogs, alterations in thepolyamine methylene backbone should have significant impact on thecompound's biological properties. In fact, the significance of chargeand the length of the methylene bridges separating the cations in thebiological properties of the polyamine analogs has been demonstrated.

Among the most preferred polyamines of the invention are those of thefollowing formula:

R₁—N¹H—(CH₂)_(m)—N²H—(CH₂)_(n)—N³H—R₂

wherein: R₁ and R₂ may be the same or different and are H or alkyl;preferably having, at most, 10 carbon atoms; most preferably, methyl,ethyl and n-propyl (with the proviso that both R₁ and R₂ may not be H);and m and n may be the same or different and are 3, 4 or 5.

Exemplary of preferred polyamines of the invention are:

dimethylnorspermidine (DMNSPD)

monoethylnorspermidine (MENSPD)

diethylnorspermidine (DENSPD)

monopropylnorspermidine (MPNSPD)

dipropylnorspermidine (DPNSPD)

dimethylspermidine (DMSPD)

monoethylspermidine [(MESPD)N1]

monoethylspermidine [(MESPD)N8]

diethylspermidine (DESPD)

monopropylspermidine [(MPSPD)N1]

monopropylspermidine [(MPSPD)N8]

dipropylspermidine (DPSPD)

dimethylhomospermidine (DMHSPD)

diethylhomospermidine (DEHSPD)

monopropylhomospermidine (MPHSPD)

dipropylhomospermidine (DPHSPD)

CH₃NH(CH₂)₄NH(CH₂)₅NHCH₃ [DM(4,5)]

CH₃CH₂NH(CH₂)₄NH(CH₂)₅NHCH₂CH₃ [DE(4,5)]

CH₃(CH₂)₂NH(CH₂)₄NH(CH₂)₅NH(CH₂)₂CH₃ [DP(4, 5)]

CH₃NH(CH₂)₅NH(CH₂)₅NHCH₃ [DM(5,5)]

CH₃CH₂NH(CH₂)₅NH(CH₂)₅NHCH₂CH₃ [DE(5,5)]

CH₃(CH₂)₂NH(CH₂)₅NH(CH₂)₅NH(CH₂)₂CH₃ [DP(5,5)]

It will be understood by those skilled in the art that the polyamines ofthe present invention may be employed to effect any desired biologicaleffect mediated by the polyamine biosynthetic network or system, e.g.,anti-neoplastic, anti-viral, anti-psoriasis, anti-inflammatory,anti-arrhythmic, etc.

For the purposes of a detailed description of a preferred embodiment ofthe invention, however, the activity of a representative number ofpolyamines against tumor cells sensitive thereto will be described.

The triamines of the invention described hereinbelow can be envisionedas belonging to one of two families of polyamines having the structuralformula:

One family of polyamines can be characterized as having symmetricalmethylene backbones, i.e., wherein m=n.

The other family is unsymmetrical, i.e., m≠n.

Synthesis of Triamines. The two families of triamines were synthesized:(1) those with symmetrical methylene backbones, i.e., derived from theparent polyamines norspermidine (3,3), homospermidine (4,4) or thelonger triamine (5,5) [wherein (3,3), (4,4) and (5,5) refer to thenumber of methylene groups, i.e., (m,n)], with an alkyl group at one orboth terminal nitrogens; and (2) those with unsymmetrical methylenebackbones, i.e., from the parent polyamines spermidine (3,4) or the(4,5) triamine, with an alkyl group at one or both terminal nitrogens(Table 1). The numbers in parentheses refer to the number of methylenesseparating successive nitrogens. In the case of theN^(α),N^(ω)-disubstituted norspermidine (m=3, n=3) and spermidine (m=3,n=4) analogues, the commercially available triamines norspermidine(NSPD) (1) and spermidine (SPD) (7) were reacted with mesitylenesulfonylchloride (3 equiv) under biphasic conditions (CH₂Cl₂/dilute NaOH) togive 30 [Bergeron et al, Drug Metab. Dispos., Vol. 23, supra], and 31,respectively (step e) (FIG. 1, Scheme 1). These trisulfonamides weredeprotonated with NaH in DMF and treated with an excess of theappropriate primary alkyl iodide to make intermediates 43, 44 and 46-49(step f). Finally, the mesitylenesulfonyl blocking groups were cleanlyremoved under reductive conditions utilizing 30% HBr in HOAc and phenolin CH₂Cl₂ (step g) to give terminal dimethyl-(2, 8), diethyl-(4, 11),and dipropyl-(6, 14) NSPD and SPD, respectively, which were isolated astheir recrystallized trihydrochloride salts.

The symmetrical triamines homospermidine (HSPD) (15) (4,4) and1,7,13-triazatridecane (24) (5,5), which were not commerciallyavailable, and their terminally dialkylated derivatives were synthesizedby a segmented synthesis (FIG. 1, Scheme 1). Mesitylenesulfonamide (35)[Bergeron et al, Drug Metab. Dispos., Vol. 23, supra] was dialkylatedwith either N-(4-bromobutyl)phthalimide to give 37 (step i) or with5-chlorovaleronitrile to furnish 39 (step c). Hydrogenation of the cyanogroups of 39 with Raney nickel in methanolic ammonia gaveN,N-bis(5-aminopentyl)mesitylenesulfonamide (42) (step d), whichprovided 5,5-triamine 24 in good yield by treatment with 30% HBr in HOAc(step g). Use of the aromatic imide blocking group in 37 avoided thesolubility problems during attempted hydrogenation (Raney nickel,methanolic NH₃) of N,N-bis(3-cyanopropyl)mesitylenesulfonamide.Hydrazinolysis of 37 in refluxing EtOH (step j) led toN,N-bis(4-aminobutyl)mesitylenesulfonamide (40). HSPD (15) itselfresulted from reductive deprotection of monosulfonamide 40 (step g).Terminal diamines 40 and 42 were converted to theirmesitylenesulfonamides 32 and 34, respectively, (step e) and werealkylated with the appropriate primary halide (step f). Hydrogenbromide-promoted deprotection of masked analogues 50, 52 and 55-57yielded DMHSPD (16), DPHSPD (19), DM(5,5) (25), DE(5,5) (26) and DP(5,5)(27), respectively.

N¹,N⁹-Diethylhomospermidine (DEHSPD) (17) was made by a convergent route(FIG. 1, Scheme 1). Alkylation of sulfonamide 35 withN-(4-bromobutyl)-N-ethylmesitylenesulfonamide (58) (Bergeron et al, J.Med. Chem., Vol. 37, supra] (2 equiv) led to triprotected analogue 51(step h), which was unmasked with HBr/HOAc, giving DEHSPD (17) (step g).

3,8,14-Triazahexadecane [DE(4,5)] (22), the terminally diethylatedanalogue of the unsymmetrical 4,5-triamine, was assembled fromN-(tert-butoxycarbonyl)-N-mesitylenesulfonamide (59), a diprotectedammonia synthon [Bergeron et al, J. Med. Chem., Vol. 37, supra] (FIG. 2,Scheme 2). Alkylation of reagent 59 withN-(4-bromobutyl)-N-ethylsulfon-amide (58) (NaH/DMF) (step a) gavetriprotected monoethylputrescine 62. The BOC group of 62 was removedwith trifluoroacetic acid (TFA) (step c). The resulting sulfonamide 63was alkylated with N-(5-bromopentyl)-N-ethylmesitylenesulfonamide (61)(step a), which was made from ethylsulfonamide 60 and excess1,5-dibromopentane (NaH/DMF) (step b), to generate fully protectedtriamine 64. Deprotection of the amino groups of 64 with HBr led to thediethylated analogue 22 (step d).

The 4,5-triamine 1,6,12-triazadodecane (20) and its dialkylatedanalogues 2,7,13-triazatetradecane [DM(4,5)] (21) and4,9,15-triazaoctadecane [DP(4,5)] (23) were produced by a segmentedsynthesis (FIG. 1, Scheme 1). Consecutive mono-alkylation of sulfonamide35 with 4-bromobutyronitrile (step a) and 5-bromovaleronitrile (step b)generated dinitrile 38. The cyano groups of 38 were reduced in a Parrshaker with Raney nickel in methanolic ammonia (step d), resulting inprimary amine 41. Cleavage of the sulfonyl group of 41 with HBr (step g)produced the parent 4,5-triamine 20. Treatment of 41 withmesitylenesulfonyl chloride (2 equiv) gave 33 (step e), which wasterminally dialkylated with iodomethane to 53 or with 1-iodopropane to54 (step f). Unmasking the amino groups led to dimethylated anddipropylated 4,5-analogues 21 and 23, respectively (step g).

N¹-Propylnorspermidine (MPNSPD) (5) was made by treatingtrimesitylenesulfonyl NSPD 30 [Bergeron et al, Drug Metab. Dispos., Vol.23, supra] with 1-iodopropane (1 equiv/NaH/DMF), and isolating 45 fromthe statistical mixture of mono- and di-alkylated products by flashcolumn chromatography (step f) (FIG. 1, Scheme 1). Since SPD isunsymmetrical, reaction of its trisulfonamide 31 with a primary alkyliodide (1 equiv) would lead to N¹- and N⁸-monoalkylated products, whichmay be difficult to separate. Thus, the synthesis of both SPD and theHSPD monopropyl analogues required a fragment synthesis (FIG. 3, Scheme3). N-Propylmesitylenesulfonamide (65) was converted to 3-bromopropyl 66or 4-bromobutyl reagent 67, with the required dibromoalkane in excess(NaH/DMF). Triphenylmethyl chloride was stirred at room temperature witheither 1,3-diaminopropane or 1,4-diaminobutane (5 equiv) in CH₂Cl₂ (stepc), resulting in N¹-tritylated-trimethylenediamine 68 or -putrescine 69.Sulfonation of 68 and 69 occurred at the primary nitrogen and not nextto the bulky triphenylmethyl group to give N,N′-disubstituted diamines70 and 71, respectively (step a). Reaction of the anions of 70 or 71with the appropriate bromide 66 or 67 resulted in regiospecificN-alkylation at the sulfonamide terminus. Specifically, reaction of 70with 67 gave 73, and 71 plus 66 or 67 led to 72 or 74, respectively. Theprotecting groups of 45 and 72-74 were removed simultaneously with Hbrin HOAc/PhOH, resulting in MPNSPD (5), MPSPD(N¹) (12), MPSPD(N⁸) (13)and MPHSPD (18), respectively.

Both N¹-(9) and N⁸-ethylspermidine (10) were obtained from reduction ofthe requisite monoacetylspermidine with lithium aluminum hydride in hotTHF, thus completing the synthesis of the triamine series.

Tetraamine analogue N¹,N¹¹-dipropylnorspermine (DPNSPM) (28) wasaccessed from commercially available norspermine (FIG. 4, Scheme 4).Bis-alkylation of the tetrasulfonamide dianion of 75 [Bergeron et al, J.Med. Chem., Vol. 37, supra] with 1-iodopropane (step a) and facileremoval of the mesitylenesulfonyl blocking groups of 76 with HBr (stepb) generated DPNSPM (28).

The longer polyamine 3,9,14,20-tetraazadocosane [DE(5,4,5)] (29), theterminally diethylated derivative of the unknown (5,4,5) tetraamine, wassynthesized in three high yield steps by the segmenting method (FIG. 5,Scheme 5). N-Ethylmesitylenesulfonamide (60) [Schreinemakers, Recl.Trav. Chim. Pays-Bas Belg., Vol. 16, pages 411-424 (1897)] wasdeprotonated (NaH/DMF) and treated with 1,5-dichloropentane (10 equiv),resulting in alkyl chloride 77 (step a).N¹,N⁴-Bis(mesitylenesulfonyl)putrescine (78) [Bergeron et al, J. Med.Chem., Vol. 37, supra] was alkylated with synthon 77 to give maskedtetraamine 79 (step b). The four blocking groups were removed with HBr(step c) to furnish DE(5,4,5) 29 as its crystalline tetrahydrochloridesalt.

Biological Evaluations. In summarizing the biological properties of thepolyamine analogues, the results are separated into three sets ofmeasurements: the 48- and 96-hour IC₅₀ values against L1210 cells andthe corresponding K values for the polyamine transport apparatus (Table1); the effect on polyamine pools (Table 2); and the impact on ODC,AdoMetDC and SSAT (Table 3). The compounds are arranged in sets byincreasing length, e.g., norspermidine, spermidine, homospermidine,(4,5)- and (5,5)-triamines. Each set is ordered in terms of the size ofthe terminal alkyl groups. While the IC₅₀ and K_(i) values of DESPD andits impact on polyamine pools, ODC, AdoMetDC and SSAT have beenpreviously reported [Porter et al, Cancer Res., Vol. 45, supra], themeasurements on this compound were repeated so that the appropriatepositive control and not a historical control would be in place. Inorder to showcase the importance of the polyamine's overall chain lengthin structure-activity relationships, there is included a briefcommentary of results of tetraamine analogues [Bergeron et al, J. Med.Chem., Vol. 37, supra] where available. Thus, numbers included inparentheses in the tables represent the values for the correspondingtetraamine analogues. A brief discussion is also presented on themetabolic profile of the triamines and on the cationic conservation ofcharge the cell maintains as defined by the polyamines. Finally, acomparison of the acute and chronic in vivo toxicities of several keytriamines and tetraamines is presented.

Antiproliferative Activity—IC₅₀ of L1210 cells. As shown in Table 1,NSPD is the most active among the NSPD family of analogues with an IC₅₀of 0.9 μM at 48 hours and 0.5 μM at 96 hours. This activity is probablyrelated to the fact that this triamine can easily be converted to toxicmetabolites [Alarcon et al, “Evidence for the Formation of CytotoxicAldehyde Acrolein from Enzymatically Oxidized Spermine or Spermidine,”Arch. Biochem. Biophys., Vol. 137, pages 365-372 (1970)]. All of thealkylated norspermidine analogues have IC₅₀ values >100 μM at 48 hours.At 96 hours, the IC₅₀ values range from 3.5 to >100 μM with an order ofDMNSPD<DENSPD<DPNSPD<MENSPD and MPNSPD (most to least active). Thus, inthis family, terminal dialkylation with smaller groups increases thecompound's activity, while triamines with a single alkyl group are lessactive than the corresponding compound with bis N^(α),N^(ω)-alkylsubstitution. In contrast, analogues of the tetraamine norspermine,although also inactive at 48 hours, were more active than thecorresponding triamines at 96 hours. Moreover, whether norspermine wassymmetrically substituted with methyl or ethyl groups or had a singleethyl fixed to one of the terminal nitrogens was insignificant relativeto the 96-hour IC₅₀ values, which were around 2 μM.

At 48 hours, SPD and all of its analogues had an IC₅₀ of at least 100μM. Unlike NSPD, and not surprisingly, SPD is the least active compoundin its family with IC₅₀ values above 100 μM at both 48 and 96 hours. At96 hours, DMSPD and DESPD are substantially more active than DPSPD. Whenan ethyl group was removed from either end of DESPD, a monoalkylatedanalogue was produced with lower activity than DESPD, by one to twoorders of magnitude. It is interesting that monoalkylation of SPD byethyl or propyl at different ends result in very different activities.At 96 hours, with an IC₅₀ of 4 μM, MESPD(N¹) was about 10 times moreactive than MESPD(N⁸). The same trend was found, although to a lesserdegree, among the two monopropyl SPD analogues in that MPSPD(N¹) wasmore than twice as active as MPSPD(N⁸). Thus, alkylation at the N¹position results in a higher activity than alkylation at N⁸ (Table 1).The spermine analogues had a significant effect on cell growth even at48 hours and at 96 hours, the IC₅₀ concentrations of tetraamines rangedfrom 0.2 to 0.8 μM, with DESPM<DPSPM<MESPM<DMSPM. Again, in everyinstance, the tetraamines were more active. It is interesting that 3.7%of intracellular N¹-MESPD and 6.1% of intracellular N¹-MPSPD aremetabolically converted to the corresponding tetraamines, ME-[3,4,3] andMP-[3,4,3] respectively (Table 4). Given the potent antiproliferativeactivity of the tetraamines in general, this may help explain theenhanced activity of the N¹-alkylspermidines in comparison to theN8-alkylspermidines since the latter are not metabolically converted totetraamines.

Among the HSPD analogues, DEHSPD is active at 48 hours with an IC₅₀ of25 μM. Other analogues' IC₅₀s are at least 100 μM at 48 hours. At 96hours, all of the IC₅₀s fall into the range from 0.3˜0.9 μM, except forDPHSPD which has an IC₅₀ of 6 μM. Compared to the norspermidine andspermidine analogues, the homospermidine analogues as a group are moreactive. With the tetraamines, the most notable differences in activitywere between the diethyl and dimethyl compounds (Table 1).

The (4,5) series are the most effective triamines identified. As thetriamine chain increases in length from (4,5) to (5,5), the activitydecreases at both 48 hours and 96 hours. Specifically, DM(4,5) andDE(4,5) have IC₅₀ values in the 2-6 μM range, while the DP(4,5) has anIC₅₀ around 100 μM at 48 hours. At 96 hours, the IC₅₀ values of bothseries substantially decrease; even DP(4,5) has an IC₅₀<2 μM. Thenumbers are uniformly higher for the (5,5) triamines even at 96 hours.The corresponding tetraamine analogues DE(4,5,4) and DE(5,4,5) are moreactive at both 48 and 96 hours.

Competitive Uptake Determinations in L1210 cells. The ability of thenorspermidines, spermidines, homospermidines, 4,5- and 5,5-triamines tocompete with radiolabeled SPD for uptake was evaluated (Table 1). Thegeneral trend is that the terminally alkylated triamines have higherK_(i) values than the unalkylated triamines and are thus less easilytaken up by the cell. In the dialkylated series of spermidines,homospermidines and 4,5-triamines, K_(i) values increase as the size ofthe terminal group increases. This is not completely true with thenorspermidines and the 5,5 triamines. The relationship holds with methyland ethyl but not for the propyl of the latter two systems. Finally, thenumber of methylenes separating the amines plays a role in determiningpolyamine uptake properties. In general the effectiveness with which theanalogues compete for uptake is spermidines≈homospermidines>4,5triamines>5,5 triamines>norspermidine. Interestingly, this same trend isobserved with the ethylated tetraamines,spermines≈homospermines>DE(3,4,4)≈DE(4,5,4)>norspermine.

TABLE 1 TRIAMINE ANALOGUE STRUCTURES, ABBREVIATIONS, L1210 GROWTHINHIBITION AND TRANSPORT IC₅₀ (μM) Abbrevia- 48 96 Structure tion hourhour K_(i (μM)) Norspermidines 1

NSPD 0.9 0.5 7.2 2

DMNSPD >100 (>100) 3.5-6.0 (2.5) 60 (5.6) 3

MENSPD >100 (>100) >100 (2.5) 34 (7.7) 4

DENSPD >100 (>100) 10 (2) 250 (17) 5

MPNSPD >100 ˜100 33 6

DPNSPD >100 (>100) 60 (18) 125 (11) Spermidine 7

SPD >100 >100 2.2 8

DMSPD >100 (>100) 1.5-1.8 (0.75) 5.1 (1.1) 9

MESPD(N1) >100 (99) 3.0-5.0 (0.33) 8.6 (1.7) 10

MESPD(N8) >100 40 7 11

DESPD ˜100 (30) 0.6-0.8 (0.18) 19.3 (1.6) 12

MPSPD(N1) >100 20-35 3.0 13

MPSPD(N8) >100 50-60 8.5 14

DPSPD >100 (3) 30-35 (0.2) 25.6 (2.3) Homospermidines 15

HSPD >100 1.7-4.0 3.4 16

DMHSPD >100 (>100) 0.9 (0.32) 5.5 (0.97) 17

DEHSPD 18-25 (0.2) 0.3-0.4 (0.07) 19 (1.4) 18

MPHSPD 100 0.5-0.7 5.0 19

DPHSPD >100 6.0 67 4,5-triamines 20

4,5-Triamine >100 0.15- 0.20 1.4 21

DM(4,5) 2.0 0.11- 0.12 21 22

DE(4,5) 3.0-6.0 (0.3) 0.19- 0.2 (0.035) 64 (6.0) 23

DP(4,5) ˜100 1.0-1.4 75 5,5-triamines 24

5,5-Triamine ˜100 0.3-0.5 13.8 25

DM(5,5) 15 0.4 133 26

DE(5,5) 10-12 (0.4) 0.65- 0.7 (0.03) 174 (16) 27

DP(5,5) >100 6.0 87

K_(i) values and IC₅₀ concentrations at 48 and 96 hours. K_(i)determinations were made by following analogue inhibition of spermidinetransport. The IC₅₀ and K_(i) values of corresponding tetraamineanalogues are shown in parentheses. The tetraamine corresponding toDE(4,5) (22) is DE(4,5,4), and the tetraamine corresponding to DE(5,5)(26) is DE(5,4,5) (29).

Polyamine Pools. The following guidelines were adopted for studying theimpact of the analogues on polyamine pools (Table 2). The measurementswere made after a 48-hour exposure to the analogue, and two differentconcentrations of analogue were evaluated in each case. For analogueswhose IC₅₀ concentration exceeded 100 μM at 48 hours, the polyaminepools were determined at 100 and 500 μM. For the other analogues, theeffect on polyamine pools was evaluated at the 48-hour IC₅₀concentration and at 5 times this number.

At 500 μM, the effects of DMNSPD, DENSPD and MPNSPD on polyamine poolswere similar (Table 2), i.e., PUT was depleted below detectable limitsand spermidine was reduced to 6-15% of controls, while spermine levelswere diminished to below 50%. DPNSPD was not as effective as the othernorspermidine analogues in depletion of polyamine pools, e.g., at 500μM, PUT was only lowered to 68%, SPD to 71% and no effect on SPM level.The dipropyl analogue was similar in behavior to the parentnorspermidine. The corresponding norspermines were again more effective.At 100 μM, the effect of DMNSPM and DENSPM on polyamine pools wassimilar, i.e., putrescine was depleted to below detectable limits andspermidine was reduced to around 5% of controls, while spermine levelswere diminished to 27-36%.

DMSPD and DESPD at 100 μM depleted PUT to below detectable limits, SPDto 5%, SPM to 58% and 74% of control, respectively. The monoalkylatedSPD analogues MESPD(N¹), MESPD(N⁸) and MPSPD(N¹) gave a similar patternof polyamine pool depletion. At 100 μM, putrescine was depleted to belowdetectable levels, spermidine to 25% and spermine to 80%, 84% and 90% ofcontrol value. MPSPD(N⁸) was slightly less active than MPSPD(N¹). At 500μM, DPSPD reduced PUT to below detectable level and SPD to around 10% ofcontrol. Like DPNSPD, DPSPD showed little suppression of SPM levels andpossibly even some up-regulation at 100 μM. Interestingly, at the levelof PUT and SPD suppression, MPSPD (N¹) and MPSPD (N⁸) behave very muchlike their MESPD counterparts. However, the propyl analogues areslightly less effective at spermine suppression. The parent amine SPDsuppresses PUT, but not SPD or SPM. Again, the corresponding sperminesare more effective than the triamines. At 100 or 500 μM DMSPM or MESPMor at 30 and 150 μM DESPM, putrescine was reduced to below detectablelimits, spermidine diminished to under 2% of control and spermine tounder 25%. At 3 μM, DPSPM reduced putrescine to below detection andspermidine to 18%, while the spermine level remained at 64% of control.At 15 μM DPSPM, spermidine was further reduced to 9% and spermine to43%.

Among the homospermidine analogues, the parent triamine, HSPD, was themost active at polyamine suppression. At 100 μM, PUT was depleted toagain below detectable levels, SPD to 4% and SPM to 32%. With all of theHSPD analogues, at 500 μM, the level of putrescine was diminished tobelow detectable limits and the SPD level below 10% of control. DMHSPDand DEHSPD had little impact on SPM level, while MPHSPD produced a milddecrease. In the case of cells grown in 100 μM and 500 μM DPHSPD, thelevel of SPM seemed to be increased compared to the controls. As isusual, the homospermines were more effective than the correspondingtriamine counterparts. At 100 μM, the homospermine analogue DMHSPM wassimilar to the corresponding alkyl spermine in its ability to depletethe polyamines. However, DEHSPM was somewhat less effective atsuppressing spermine pools in comparison to DESPM.

Similar results were observed with homospermidine homologues, the (4,5)and (5,5) triamines. At 500 μM, the (4,5) and (5,5) parent aminesdepleted both PUT and SPD below detectable level and SPM to 35% and 20%of control, respectively. DM(4,5) at 10 μM and DE(4,5) at 15 μM reducePUT below detectable limits and SPD to 18% of control. However, neitheris very effective at reducing SPM levels. DP(4,5) even at 500 μM, whileit depletes the cell of PUT, only reduces SPD to 31% of control withpossible stimulation of SPM. Finally, DP(5,5) is only marginally active,requiring a 500 μM concentration to even reduce PUT by 50% and SPD by30% and with no impact on SPM. However, the homospermine homologuesDE(4,5,4) and DE(5,4,5), both of which demonstrated low 48-hour IC₅₀values, 0.3 and 0.4 μM, respectively, were similar to the correspondingtriamines at reducing polyamines.

TABLE 2 IMPACT OF TRIAMINE ANALOGUES ON POLYAMINE POOLS^(a) Conc. Compd(μM) Put Spd Spm Analogue^(b) Norspermidines 1 NSPD 0.9 38 44 113 1.0945 0 12 83 2.14 2 DMNSPD 100(100) 0(0) 9(5) 58(36) 5.00(2.14) 500(500)0(0) 6(3) 48(27) 5.51(1.84) 4 DENSPD 100 (10) 0(30) 17(14) 74(31)3.67(1.59) 500(100) 0(0) 7(6) 47(30) 3.77(2.44) 5 MPNSPD 100 0 29 563.07 500 0 15 49 4.78 6 DPNSPD 100(100) 70(61) 76(35) 96(77) 0.49(0.89)500(500) 68(0) 71(19) 102(56) 1.24(1.28) Spermidines 7 SPD 100 0 117 118— 500 0 145 108 — 8 DMSPD 100(100) 0(0) 5(0) 58(21) 4.96(1.26) 500(500)0(0) 0(0) 54(24) 4.89(1.24) 9 MESPD(N1) 100(160) 0(0) 25(1) 80(21)4.20(1.24) 500(500) 0(0) 14(1) 53(19) 4.73(1.23) 10 MESPD(N8) 100 0 2684 4.41 500 0 15 61 4.96 11 DESPD 100(30) 0(0) 5(0) 74(12) 4.61(0.40)500(150) 0(0) 0(0) 55(14) 4.20(1.13) 12 MPSPD(N1) 100 0 25 90 3.97 500 015 72 4.95 13 MPSPD(N8) 100 0 33 103 3.52 500 0 16 95 5.16 14 DPSPD100(3) 6(0) 35(18) 135(64) 3.26(1.12) 500(15) 0(0) 12(9) 99(43)3.69(1.09) Homospermidines 15 HSPD 100 0 4 32 3.58 500 0 2 21 4.44 16DMHSPD 100(100) 0(0) 3(0) 106(30) 5.51(1.49) 500(500) 0(0) 0(0) 106(27)5.85(1.03) 17 DEHSPD 25(10) 0(0) 6(0) 114(61) 4.61(2.94) 125 0 3 97 4.6918 MPHSPD 100 0 2 82 5.16 500 0 0 66 5.86 19 DPHSPD 100 0 19 144 3.12500 0 7 111 3.68 4,5-Triamines 20 4,5 100 0 1 53 3.02 500 0 0 35 3.20 21DM(4,5) 2 0 33 112 2.79 10 0 18 111 5.36 22 DE(4,5) 3(0.3) 0(44) 47(61)99(70) 1.20(0.26) 15(1.5) 0(0) 18(5) 98(31) 3.40(0.72) 23 DP(4,5) 100 040 119 1.40 500 0 31 121 2.42 5,5-Triamines 24 5,5 100 0 0 33 2.58 500 00 20 2.50 25 DM(5,5) 15 0 33 115 2.56 75 0 20 101 3.61 26 DE(5,5)15(0.15) 0(37) 55(55) 97(88) 1.09(0.34) 75(0.75) 0(0) 23(10) 73(58)1.59(1.48) 27 DP(5,5) 100 59 73 97 0.86 500 51 69 103 1.33

^(a) Putrescine (Put), spermidine (Spd) and spermine (Spm) Levels after48 hours of treatment are given as % polyamine found in untreatedcontrols. Typical control values in pmol/10⁶ L1210 cells are Put=260±59,Spd 3354±361, Spm=658±119.

^(b) Analogue amount is expressed as nmol/10⁶ cells. Untreated L1210cells (10⁶) correspond to about 1 μL volume; therefore, concentrationcan be estimated as nmol/mM.

Impact of Analogues on ODC and AdoMetDC Activities. A comparison of theeffects of the triamine versus tetraamine polyamine analogues on ODC andAdoMetDC clearly demonstrates that the tetraamines are more effective atsuppressing these enzymes than the corresponding triamines [Bergeron etal, J. Med. Chem., Vol. 37, supra]. Previous studies [Porter et al,“Regulation of Ornithine Decarboxylase Activity by Spermidine and theSpermine Analogue N¹,N⁸-Bis(ethyl)spermidine,” Biochem. J., Vol. 242,pages 433-440 (1987); and Porter et al, “Combined Regulation ofOrnithine and S-Adenosylmethionine Decarboxylase by Spermine and theSpermine Analogue N¹,N²-Bis(ethyl)-spermine,” Biochem. J., Vol. 268,pages 207-212 (1990)] suggested that the effect of the polyamineanalogues on ODC and AdoMetDC is fairly rapid. For example, DESPMinduced reduction in ODC activity plateaued at 4 hours and AdoMetDC at 6hours. On the basis of these studies, it was elected to evaluate theimpact of the triamines on ODC and AdoMetDC at 4 and 6 hours,respectively.

The parent triamine norspermidine reduced ODC activity to 11% ofcontrol, the corresponding dimethyl, DMSPD, to 17%, the diethylanalogue, DENSPD, to 80% and the dipropyl compound, DPNSPD, had noeffect on this enzyme (Table 3). Monoethylnorspermidine, MENSPD, wasmore active than the corresponding dialkyl analogue, DENSPD, withreduction to 42 versus 80% of control, as was the monopropyl, MPNSPD,relative to its dipropyl counterpart DPNSPD, with reduction to 33 versus100% of control. In 4 hours, 1 μM DMNSPM, MENSPM or DENSPM reduced ODCactivity to nearly the same extent, to approximately 7% of control. Thetriamines are generally less effective than the correspondingtetraamines at suppressing AdoMetDC, although the differences are not asprofound with the norspermidines versus the norspermines. Thenorspermidines reduce the AdoMetDC to 41-58% of control and thenorspermines to 33-49% of control, except the dipropyls, which are atbest marginally effective.

The spermidine analogues are less active than the correspondingspermines but more effective at reducing ODC activity than thenorspermidines. At 1 μM DMSPM, MESPM or DESPM, ODC activity was reducedto 10% or less of control, while ODC in DPSPM-treated cells was onlylowered to 52% of controls. The parent triamine spermidine reduces ODCto 16% of control while the alkylated analogues except for DPSPD,diminish ODC activity to between 10-30% of control. Again, the monoalkylanalogues are more effective than the corresponding dialkyl compounds.MESPD(N¹) and MESPD(N⁸) reduce ODC to 10 and 17% versus 30% for DESPD.This property of the monoalkylated analogue is even further accentuatedwith the propylated spermidines MPSPD(N¹) and MPSPD(N⁸) versus DPSPDlowering ODC to 18 and 14% versus 75% of control. Again, when comparingdialkylated compounds, the larger the alkyl substituent the less activethe analogue.

The spermidine analogues were less effective than the correspondingnorspermidines and spermines at reducing AdoMet activity. The spermidineanalogues, except for DPSPD, reduce AdoMetDC to under 70% of controlactivity. DPSPD has no impact on the enzyme. Again, as with ODC, themonoalkylated spermidine compounds were generally more active than thedialkylated compounds. DMSPM, MESPM or DESPM at 1 μM almost paralleledthe ability of the corresponding norspermine analogues to suppressAdoMetDC with an average reduction to 33% of control, slightly moreactive than the spermidines. DPSPM at 1 μM reduced AdoMetDC activity to72% of that seen in untreated cells.

The homospermidines were less active than the correspondinghomospermines at reducing ODC activity, but similar in behavior to thespermidines. Also, consistent with the norspermidine and spermidineresults, the triamines with the larger substituent, propyl, were leasteffective and the monoalkyl compounds were more active than thecorresponding dialkyl ones. Finally, the homospermidine analogues,except for MPHSPD, were not effective at AdoMetDC inhibition andcertainly less active than the corresponding tetraamines.

Interestingly, adding a methylene unit to DEHSPM to produce DE(4,5,4)resulted in a decrease in ODC suppressing activity. ODC was lowered toonly 7% of control with DEHSPM and to 20% of control with DE(4,5,4).This methylene addition had little effect on reduction of AdoMetDCactivity, to about 40% for both. The same phenomenon was observed onmoving from lower alkylhomospermidines to the dialkyl (4,5) and dialkyl(5,5) compounds, the ODC-suppressing capacity substantially decreasedwhile the AdoMetDC properties were similar to those of thehomospermidines. It is noteworthy, however, that the parent(4,5)-triamine demonstrates reasonably effective suppression of ODC andAdoMetDC. The (5,5) parent triamine is an effective and highly selectiveODC antagonist, reducing ODC to 16% of control with little effect onAdoMetDC. Other than this, there is little effect on either ODC orAdoMetDC by (5,5) analogues. The tetraamine analogue DE(5,4,5) is farmore active against both ODC and AdoMetDC than DE(5,5).

Impact of Triamine Analogues on SSAT Activity. While the influence ofchain length and terminal substituents are more monotonic regardingtheir effect on the analogues' suppression of ODC and AdoMetDC, thereare nevertheless some notable structure-activity relationships for SSATstimulation. The ability of triamine analogues to up-regulate SSAT inL1210 cells is remarkably sensitive to small structural changes (Table3; FIG. 6a). For example, the diethyl triamines stimulate SSAT 780% forDE(3,3) to a peak of 1380% for DE(3,4), with a decrease to 640% forDE(4,4) and falling to essentially control values for DE(4,5), 120%, andDE(5,5), 90%. The DE triamine structure activity curve appears to beshifted to the right from the corresponding DE tetraamine curve (FIG.6b). Thus, the DE tetraamine curve is maximal at 1500% of control forDE(3,3,3) and falls to nearly control value for DE(4,4,4), DE(4,5,4) andDE(5,4,5).

Substituent changes on the triamines have a profound effect on SSATstimulation only with the (3,3) and (3,4) compounds. The differences aremore compressed for the (4,4) and (4,5) triamines and absent with (5,5)triamines. In the case of the tetraamines, the (3,3,3) system is theonly framework in which a marked effect in SSAT stimulation is observedwith substituent changes. While there are some changes with the (3,4,3)backbone, these are again compressed.

With both the triamines and tetraamines, unlike with ODC and AdoMetDC,there is no relationship between substituent size and SSATup-regulation. However, when clear differences exist between stimulatoryabilities, i.e., (3,3), (3,4), (3,3,3), the ethyl group is clearly thesuperior.

TABLE 3 EFFECT OF POLYAMINE HOMOLOGUES ON ORNITHINE DECARBOXYLASE (ODC),S-ADENOSYLMETHIONINE DECARBOXYLASE (AdoMetDC) AND SPERMIDINE/SPERMINEACETYLTRANSFERASE (SSAT) IN L1210 CELLS Compd ODC AdoMetDC SSATNorspermidines 1 NSPD 11 62 150 2 DMNSPD 17(6) 41(49) 250(200) 3 MENSPD42(5) 58(33) 390(410) 4 DENSPD 80(10) 45(42) 780(1500) 5 MPNSPD 33 38470 6 DPNSPD 100(79) 99(70) 220(460) Spermidines 7 SPD 16 43 160 8 DMSPD22(3) 68(40) 270(300) 9 MESPD(N1) 10(10) 58(27) 430(150) 10 MESPD(N8) 1754 400 11 DESPD 30(3) 68(28) 1380(460) 12 MPSPD(N1) 18 56 1200 13MPSPD(N8) 14 64 500 14 DPSPD 75(52) 107(72) 1030(500) Homospermidines 15HSPD 11 54 430 16 DMHSPD 20(4) 86(45) 510(140) 17 DEHSPD 47(7) 90(41)640(110) 18 MPHSPD 20 59 570 19 DPHSPD 86 123 420 4,5-triamines 204,5-triamine 19 57 410 21 DM(4,5) 56 71 130 22 DE(4,5) 100(20) 70(39)120(120) 23 DP(4,5) 83 86 80 5,5,triamines 24 5,5-triamine 16 88 90 25DM(5,5) 105 97 90 26 DE(5,5) 100(19) 109(54) 90(190) 27 DP(5,5) 73 12390

Enzyme activity is expressed as percent of untreated control for ODC (1μM at 4 hours), AdoMetDC (1 μM at 6 hours) and SSAT (10 μM at 48 hoursfor triamine analogues, and 2 μM at 48 hours for tetraamine analogues).The ODC, AdoMetDC and SSAT levels of corresponding tetraamine analoguesare shown in parentheses.

Metabolism. In an experiment focused on the impact of DPNSPD (DP-[3,3]in Table 4) on polyamine pools, a substantial unexpected peak appearedin the chromatogram of treated cells. The suspicious peak was shown tocorrespond to MPNSPD (MP-[3,3] in Table 4) as confirmed by co-elutionwith an authentic sample. The intracellular levels of MPNSPD after a48-hour exposure to DPNSPD was about 50% of intracellular level of theparent compound. Although N-dealkylation had been shown to be animportant step in the metabolism of the alkylated tetraamines DENSPM[Bergeron et al, Drug Metab. Dispos., Vol. 23, supra] and DEHSPM[Bergeron et al, “Metabolism and Pharmacokinetics ofN¹,N¹⁴-Diethylhomospermine,” Drug Metab. Dispos., Vol. 24, pages 334-343(1996)] in vivo in rodents, dogs and man, previous in vitro studies withDEHSPM or DESPM [Bergeron et al, J. Med. Chem., Vol. 37, supra] in L1210cells revealed either little or no N-dealkylation under the conditionsof the experiments. The observation of N-depropylation of DPNSPDcompelled a closer look at the metabolism of the polyamine analogues inL1210 cells. In particular, the significance of the nature of theN-alkyl groups on N-dealkylation was evaluated, in addition to thelength and symmetry of the polyamine backbone.

TABLE 4 METABOLIC TRANSFORMATION OF POLYAMINE ANALOGUES BY L1210 CELLSAnalog# N-Monodealkylation {circumflex over ( )}Deaminopropylation {circumflex over ( )} Elaboration {circumflex over( )} DM-[3,3] 5000(100%) no N-demethylation (100 μM) MM-[3,3,3]2633(82.9%) no N-demethylation MM-[3,3] 523(16.5%) MM- 20(0.6%) (100 μM)[3]** DE-[3,3,3] 2440(95.3%) ME-[3,3,3] 194(4.7%) (500 μM) DE-[3,3]3761(90.7%) ME-[3,3] 194(4.7%) ME-[3] 192(4.6%) (500 μM) ME-[3,3]3051(78.2%) [3,3] 20(0.5%) ME-[3] 831(21.3%) no elaboration of (100 μM)N-Monoalkyl [3,3] DP-[3,3,3] 893(78.2%) MP-[3,3,3]* 160(14.0%) MP-[3,3]89(7.8%) (100 μM) DP-[3,3] 404(57.7%) MP-[3,3] 214(30.6%) [3, 64 MP-[3]*18(216%) (100 μM) 3]*** (9.1%) MP-[3,3] 3019(94.3%) [3,3] 144(4.5%)MP-[3]* 37(1.2%) no elaboration of (100 μM) N-Monoalkyl [3,3] DM-[3,4]5000(100%) no N-demethylation (100 μM) DE-[3,4] 4041(96.3%) N8-ME-[4,3]32(0.8%) N1-ME- 101 (100 μM) [3,4] (2.4%) N1-ME-[3,4] 3946(96.3%) [3,4]not determined ME- 150 (100 μM) [3,4,3] (3.7%) N8-ME-[4,3] 4825(99.4%)[3,4] not determined ME-[4] 29(0.6%) no elaboration of (100 μM) N8-Alkyl[4,3] DP-[3,4,3] 1568(79.0%) MP-[3,4,3]* 361(18.2%) N1-MP-[3,4] 57(2.9%)(15 μM) DP-[3,4] 3260(90.1%) N8-MP-[4,3] 192(5.3%) N1-MP- 166 (100 μM)[3,4] (4.6%) N1-MP-[3,4] 4238(93.9%) [3,4] not determined MP- 275 (100μM) [3,4,3] (6.1%) N8-MP-[4,3] 3549(99.3%) [3,4] not determined MP-[4]*26(0.7%) no elaboration of (100 μM) N8-Alkyl [4.3] DM-[4,4] 5500(100%)no N-demethylation no exposed primary aminopropyl terminal (100 μM)segment DE-[4,4,4] 4215(100%) no N-deethylation ″ (100 μM) DH-[4,4]4215(100%) no N-deethylation ″ (100 μM) DP-[4,4] 3149(87.7%) MP-[4,4]441(12.3%) ″ no elaboration of (500 μM) N-Monoalkyl [4,4] DM-[4,5]2790(100%) no N-demethylation no exposed primary aminopropyl terminal (2μM) segment DE-[4,5] 4215(100%) no N-deethylation ″ (100 μM) DP-[4,5]1325(69.7%) N1-MP- 576(30.0%) ″ no elaboration of (100 μM) [5,4]*N-Monoalkyl [5,4] DM-[5,5] 2560(100%) no N-demethylation no exposedprimary aminopropyl terminal (15 μM) segment DE-[5,5] 1585(100%) noN-deethylation ″ (75 μM) DP-[5,5] 1300(100%) no N-depropylation ″ (80μM) # L1210 cells were grown 46 hours in medium containing polyamineanalogue at the indicated concentration. Then the polyamine contents ofthe cells were analyzed by HPLC of the fluorescent DANSYL derivatives. {circumflex over ( )}Concentrations of parent drug and metabolites inL1210 cells are in pmols/10⁶ and as % of total drug in the cell. *Anauthentic sample of these presumed metabolites was not available foranalytical reference. All other metabolites were identified andquanititaed by comparison to authenticated reference compounds. **Formedby deaminopropylation of primary metabolite MM-[3,3]. ***Formed byN-depropylation of MP-[3,3].

In order to assure that the observation was not some artifact of theexperimental conditions, it was assessed whether or not components ofthe culture media itself were responsible for dealkylation (Table 5).Fetal bovine serum (FBS), for example, is well known to contain amineoxidases [Morgan, “Polyamine Oxidases and Oxidized Polyamines,” Chapter13 in The Physiology of Polyamines, Vol. I; Bachrach et al, eds., CRCPress: Boca Raton, Fla. (1989), pages 203-229]. Indeed, 1 mMaminoguanidine, an inhibitor [Gahl et al, “Reversal by Aminoguanidine ofthe Inhibition of Proliferation of Human Fibroblasts by Spermidine andSpermine,” Chem.-Biol. Interactions, Vol. 22, pages 91-98 (1978)] ofbovine serum amine oxidase present in the standard L1210 cell culturemedia, did not totally eliminate such FBS-related amine oxidaseactivity. When the “complete” RPMI-40 medium containing FBS and 1 mMaminoguanidine was incubated in the presence of 100 or 500 μM DPNSPD, asmall amount (<3%) of the DPNSPD was metabolized to MPNSPD in theabsence of L1210 cells. This corresponds to a comparatively lowextracellular concentration of MPNSPD (˜3 μM) and, given its relativelypoor affinity (K_(i)=33 μM) for the polyamine transport apparatus,argues against the extracellular medium as a major source of the highlevels of MPNSPD (264 μM) seen intracellularly. This conclusion isfurther supported by experiments which partially or totally eliminatethe source of extracellular metabolism. For example, when FBS wasreplaced with either NuSerum, a semi-synthetic substitute, or purifiedbovine serum albumin, a high level of intracellular metabolite (50% ofparent analogue, Table 5) was still observed. The chelatorbathophenanthroline disulfonic acid is a well-known inhibitor of theCu-dependent amine oxidases present in plasma [Frieden, “Complex Copperof Nature,” Metamorphosis, A Problem in Developmental Biology, 2nd ed.,Gibert et al, eds., Plenum Press: New York, N.Y., pages 478-483 (1981)]and, given its comparatively high MW and anionic charge, does not crossthe cell membrane [Alcain et al, “Iron Reverses Impermeable ChelatorInhibition of DNA Synthesis in CCl39 cells,” Proc. Natl. Acad. Sci.U.S.A., Vol. 91, pages 7903-7906 (1994); and Glahn et al,“Bathophenanthroline Disulfonic Acid and Sodium Dithionite EffectivelyRemove Surface-Bound Iron from CaCO₂ Cell Monolayers,” J. Nutr., Vol.125, pages 1833-1840 (1995)]. As expected, bathophenanthrolinedisulfonic acid completely abolished the ability of RPMI-40+10% FBS toconvert DPNSPD to MPNSPD. However, when cells were grown inRPMI-containing FBS and bathophenanthroline disulfonic acid, highintracellular concentrations of MPNSPD corresponding to ca. 50% of theintracellular DPNSPD content were observed. These results are in keepingwith the idea that the dealkylation indeed takes place within L1210cells.

TABLE 5 METABOLISM OF DPNSPD IN DIFFERENT CULTURE SYSTEMS MetabolitesAssay # Experiment Treatments (% of DPNSPD) 1 FBS^(a) + L1210 (48 hours)MPNSPD (50%) 2 NuSerum^(a) + L1210 (48 hours) MPNSPD (50%) 3 FBS (48hours) MPNSPD (3%) 4 Albumin^(b) + L1210 (4 hours) MPNSPD (50%) 5 FBS +bathophenanthroline disul- MPNSPD (0%) fonic acid (0.1 mM) (48 hours) 6FBS + L1210 + bathophenanthroline MPNSPD (50%) disulfonic acid (0.1 mM)(48 hours)

In all of the assays, RPMI-1640 was used as culture media. NuSerum IV isa semi-synthetic FBS substitute, containing 25% of FBS. ^(a) Atconcentration of 10%. ^(b) At concentration of 1.5%.

Assured that what was being seen were the results of intracellularmetabolic transformation of bisalkylated triamines, an examination ofthe influence of polyamine analogue structure on the metabolite patternobserved in L1210 cells was undertaken. These results are detailed inTable 4 for the bisalkyl triamines and a number of their primarymetabolites. Several representative tetraamine analogues are alsoincluded to demonstrate their similar metabolic fate to thecorresponding triamines. Note, too, that the structures are depictedwith the polyamine backbone described as Arabic numerals separated bycommas so that the numeral represents the number of methylenes in thelinear alkane sections separating amine centers, thus [3,3]=NSPD,[3,4,3]=SPM, [4]=putrescine, and so forth.

Three types of metabolic transformations explain the particular patternsobserved (FIG. 7). First, bisalkyl polyamines must undergoN-dealkylation before any further metabolism can occur. If thisN-dealkylation results in exposure of a primary aminopropyl segment, theprimary metabolite(s) may undergo deaminopropylation by the SSAT/PAOpolyamine degradation pathway. If this N-dealkylation results inexposure of a primary aminobutyl segment, then the triamine mightundergo elaboration into a tetraamine by serving as a substrate forspermine synthase, which anneals an aminopropyl segment derived fromS-adenosylmethionine (AdoMet) to the free aminobutyl end of themolecule. Below, the evidence as revealed in the metabolite patternsthat support these three types of metabolic transformations is detailed,along with comments on the implications these data have with respect tothe structural requirements of the corresponding enzyme systems in vivo.

A careful inspection of chromatograms from cells treated with DM-[3,3],DM-[3,4], DM-[4,4], DM-[4,5] and DM-[5,5] revealed no N-demethylation(Table 4). The dimethyl tetraamines DM-[3,3,3], DM-[3,4,3] andDM-[4,4,4] also showed no evidence of N-dealkylation (data not shown),and the unsymmetric tetraamine MM-[3,3,3] is only metabolized bydeaminopropylation at the primary amine terminus end of the molecule.

Treatment of cells with DE-[3,3] or the corresponding tetraamine,DE-[3,3,3], each resulted in the monodeethylated metabolite, ME-[3,3] orME-[3,3,3], respectively, in similar amounts: 4.7% on a mole percentbasis of the total (parent drug+identified metabolites) in the cell.Interestingly, cells treated with the unsymmetric DE-[3,4] contain eachof the two possible monodeethylated metabolites, N1-ME-[3,4] andN8-ME-[4,3] with the total amount representing about 4% of the drug inthe cell. Among the diethylated triamine analogues, only DE-[3,3] andDE-[3,4] showed N-deethylated metabolite(s); the analogues with longerbackbones, i.e., DE-[4,4], DE-[4,5] and DE-[5,5], do not showsignificant N-deethylation at all.

Of the five different dipropyl triamines which were evaluated, DP-[3,3],DP-[3,4], DP-[4,4], DP-[4,5] and DP-[5,5], all but DP-[5,5] showedsignificant N-depropylation. As suggested from the quantity of analoguepresent in cells as monodealkylated metabolite (Table 4),N-depropylation in general occurs to a greater extent thanN-deethylation. For example, in L1210 cells treated with DP-[3,3], 57.7%is present as the parent drug, DP-[3,3], 30.6% as the mono-N-dealkylatedmetabolite, MP-[3,3], 9.1% as the di-N-dealkylated metabolite, [3,3],and 2.6% as the secondary metabolite, MP-[3], formed bydeaminopropylation of MP-[3,3]. The same general pattern holds for cellstreated with the corresponding tetraamine, DP-[3,3,3], where 14.0% ofthe total is present as MP-[3,3,3] and 7.8% as MP-[3,3] formed bysecondary deaminopropylation of MP-[3,3,3]. Cells treated with thecorresponding diethyl analogues contain substantially lower amounts ofmetabolites by comparison so that 90.7% of DE-[3,3] or 95.3% ofDE-[3,3,3] is present as the unmetabolized parent compound.

The dipropyl triamine with the shortest backbone DP-[3,3] seemed mostsensitive to metabolism with MP-[3,3] representing 31% of the totaldrug. With DP-[3,4], both possible monoalkylated products N1-MP-[3,4]and N8-MP-[4,3] were detected at levels corresponding to 4.6% and 5.3%,respectively, of the total drug in the cell. Cells exposed to DP-[4,4]contained the mono-N-dealkylated metabolite, MP-[4,4], representing12.3% of the total drug in the cell. Interestingly, only one of the twopossible N-dealkylated metabolites was apparent in cells treated withthe unsymmetric triamine DP-[4,5], and this MP-[5,4] metaboliterepresented 32.9% of the total drug in the cell. When DP-[5,5] wasevaluated, no metabolic products were found, suggesting that theaminobutyl end of DP-[4,5] system was selectively dealkylated to formthe monodealkylated metabolite, N10-MP-[5,4].

If mono-N-dealkylation exposes a primary aminopropyl terminus, thiscompound is subject to further metabolism by the SSAT/PAO system presentin all cells. First, SSAT acetylates the exposed primary amine end, thenPAO oxidatively deaminates at the interior secondary amino nitrogen ofthe acetamidopropylamine segment to give acetamidopropanal, i.e., netdeaminopropylation of the substrate. PAO actively deaminopropylatesN¹-acetylspermine and N¹-acetylspermidine, the native substrates, butdoes not recognize the acetamidobutyl segment of N⁸-acetylspermidine orN-acetylputrescine as substrate. Table 4 demonstrates that, in L1210cells, there is a strict adherence to this specificity for a primaryaminopropyl segment for further metabolism of the monoalkylatedtriamines, i.e., only examples of deaminopropylation are observed. Forexample, the tetraamine MM-[3,3,3] shows a substantial amount of thedeaminopropylation metabolite, MM-[3,3], representing 16.5% of the totaldrug in the cell and even some MM-[3] (0.6%), the product ofdeaminopropylation of MM-[3,3]. No examples of deaminobutylation areseen, e.g., N8-alkyl-[4,3], monoalkyl-[4,4] and monoalkyl-[5,4] do notgive rise to such metabolites. In the case of cells treated withME-[3,3] or MP-[3,3], both N-dealkylation and deaminopropylation areavailable paths of primary metabolism. The deaminopropylationmetabolite, ME-[3], represented 21.3% of the total drug in the ME-[3,3]treated cells compared to only 0.5% for the N-deethylation product,[3,3]. In contrast, the N-depropylation product, [3,3] (4.5%),predominated compared to the deaminopropylation metabolite, MP-[3], inMP-[3,3] treated cells.

In cells treated with the N¹-monoalkylated spermidines, N1-ME-[3,4] orN1-MP-[3,4], peaks corresponding to the respective tetraaminesME-[3,4,3] (3.7% of total drug in cell) and MP-[3,4,3] (6.1% of totaldrug in cell) were observed in the HPLC chromatograms of the dansylatedcell extract (Table 4). In the case of cells containing substantialamounts of triamine analogues with a free primary aminopropyl end (i.e.,ME-[3,3], MP-[3,3], N8-ME-[4,3] and N8-MP-[4,3]), no evidence of atetraamine elaboration metabolite was observed. Only in those caseswhere a free aminobutyl end was available on a spermidine, [3,4],backbone was a tetraamine metabolite produced. No such metabolite wasproduced from triamines with a free aminobutyl end on a longer backbone(i.e., MP-[4,4] or N10-MP-[5,4]).

Thus, it is likely that at least two of the pathways responsible formetabolic transformation involve enzymes of the polyamine metaboliccycle present in all cells. Spermine synthase is responsible forelaboration of a spermidine analogue to the correspondingN-alkylspermine by annealing an aminopropyl segment to an exposedprimary aminobutyl end of the triamine. The deaminopropylation observedin L1210 cells treated with triamine and tetraamine analogues is readilyexplained as a consequence of action by the SSAT/PAO polyaminedegradative enzymes. The possibility that the N-dealkylation steprequired for further metabolic transformation of bisalkylpolyamines mayalso involve PAO is an interesting question raised by the metabolicpatterns observed.

N-Dealkylation of analogues with a hydrophobic segment shorter thanN-propyl appears to occur much less efficiently in the case of N-ethyl,or not at all in the case of N-methyl. Among the reported amineoxidases, polyamine oxidase (PAO) is the only one which usually attacksat a secondary amine center, three hydrophobic methylene carbonsinternal to the neutral N¹-acetamido nitrogen terminus ofN¹-acetylspermidine, for example. The corresponding acetamidobutylsegment of N⁸-acetylspermidine is not recognized and, therefore, notdeaminobutylated.

Conservation of Charge. In two earlier studies, it was noted that therewas a conservation of charge with respect to the total tetraaminecationic picoequivalence in the cell [Bergeron et al, Cancer Res., Vol.49, supra; and Porter et al, Cancer Res., Vol. 51, supra]. For example,if, after 24 hours of exposure to an alkylated polyamine, each of theequivalent concentrations associated with charge on the amines of boththe analogues and natural polyamine is added together, the numbers arefairly constant. For example, each picoequivalent of putrescine isassociated with two picoequivalents of cationic charge, eachpicoequivalent of spermidine or its analogues with three, and eachpicoequivalent of spermine with four. In order to maintain this balanceof charge, the cell processes the natural polyamines, e.g., exports themas it incorporates the analogues. The maintenance of total cellularcharge holds for all of the triamines examined, except the 5,5 triamines(Table 6). The implication is that the cell will not incorporate theanalogue beyond a point where the charge balance is disrupted, at whichtime cell death may occur. In the case of the tetraamines, theconservation of charge behavior seems to hold for 24 hours, but erodesafter a period of time [Bergeron et al, Cancer Res., Vol. 49, supra].With the triamines, the conservation of charge continues even at 48hours.

TABLE 6 SUMMATION OF INTRACELLULAR LEVELS OF ANALOGUES AND POLYAMINESANALYZED FOR AMINE EQUIVALENCE AFTER EXPOSURE TO POLYAMINE ANALOGUESPolyamine Picoequivaents of Amine Average ± Standard Analogues 10⁶ cells(×10³) Deviation Control Cell 13.21 2 DMNSPD 18.40 4 DENSPD 13.70 5MPNSPD 17.71 6 DPNSPD 15.01 16.21 ± 2.22 8 DMSPD 16.09 9 MESPD(N1) 16.9910 MESPD(N8) 17.99 11 DESPD 14.05 12 MPSPD(N1) 18.25 13 MPSPD(N8) 19.5914 DPSPD 15.33 16.90 ± 1.89 16 DMHSPD 20.34 17 DEHSPD 16.92 18 MPHSPD20.55 19 DPHSPD 15.99 18.45 ± 2.34 21 DM(4,5) 20.81 22 DE(4,5) 14.59 23DP(4,5) 15.15 16.85 ± 3.44 24 DM(5,5) 15.5 25 DE(5,5) 9.01 26 DP(5,5)13.91 12.81 ± 3.38 All Analogues Mean 16.47 ± 2.08

The L1210 cells were treated with polyamine analogues at 500 μM, exceptDEHSPD (125 μM), DM(4,5) (10 μM), DE(4,5) (15 μM), DM(5,5) (75 μM) andDE(5,5) (75 μM), for 48 hours. Levels of amine aquivalence for everyanalogue treated cell are averages from analysis of triplicate cellsamples. Values are obtained by multiplying the number of moles ofspermine by four, spermidine by three, putrescine by two and analogue bythree. The typical control values in nmol/miltion L1210 cells arePUT=0.260±0.059, SPD=3.354±0.361, SPM=0.658±0.119.

Acute and Chronic Toxicity of Triamines. In early studies of polyaminetoxicity in laboratory animals, triamines were found less toxic thantetraamines. Spermidine was approximately one-twentieth as nephrotoxicas spermine, and putrescine was the least toxic [Tabor et al,“Pharmacology of Spermine and Spermidine. Some Effects on Animals andBacteria,” J. Pharmacol. Exp. Ther., Vol. 116, pages 139-155 (1956); andShaw, “Some Pharmacological Properties of the Polyamine Spermine andSpermidine—a Re-appraisal,” Arch. Int. Pharmacodyn. Ther., Vol. 198,pages 36-48 (1972)].

In the current study, the acute toxicity of six analogues and thechronic toxicity of two triamines were measured (Table 7). The value ofall polyamine LD₅₀s are shown in both mg/kg and mmol/kg for comparison.For acute toxicities, the polyamine analogues were administered as asingle i.p. injection to groups of five or six animals at each dose. Theanimals were scored two hours after administration of the drug. It isclear that the acute LD₅₀s for triamine analogues are approximatelytwice the acute LD₅₀s for the corresponding tetraamine analogues.

In the chronic toxicity regimen, mice were administered the polyamineanalogue in three doses per day (t.i.d.) for six days for a total ofeighteen injections per animal and observed for 10 days after the finaldose for lethalities. The most active triamine DE(4,5) against L1210cells in vitro and the spermidine analogue DE(3,4) demonstrated muchless toxicity in mice than the related tetraamines DE(4,5,4), DE(5,4,5)and DE(3,4,3). In the early study of tetraamines, a preliminaryinvestigation suggested a direct ratio relationship between the IC₅₀ andthe chronic LD₅₀ values [Bergeron et al, J. Med. Chem., Vol. 37, supra].However, in the triamine systems, the 96-hour IC₅₀ values of DE(4,5)suggested that this triamine should be about 5 times less toxic thancorresponding tetraamine analogue, DE(4,5,4), and six times less toxicthan DE(5,4,5) (Table 1), but, in fact, they are about eight-fold lesstoxic than the DE(4,5,4) and greater than ten-fold less toxic thanDE(5,4,5) (Table 7). A similar difference is also observed in thechronic toxicity of DE(3,4). The ratio of the triamine to the tetraamine96-hour IC₅₀s suggests that DE(3,4) should be approximately four timesless toxic than DE(3,4,3), but, in fact, DE(3,4) is about five timesless toxic than DE(3,4,3) in vivo. These results suggest a potentialwidening of the therapeutic window, which renders the triamine analoguesas promising anti-neoplastics of lower toxicity and encourages furtherpursuit of animal studies.

TABLE 7 COMPARISON OP THE ACUTE AND CHRONIC TOXICITY OF TETRAAMINE ANDTRIAMINE ANALOGUES ON MICE TETRAAMINES TRIAMINES Acute^(a) LD₅₀ mg/kgChronic^(b) LD₅₀ mg/kg- Acute LD₅₀ mg/kg Chronic LD₅₀ mg/kg- Compound(mmol/kg) day (mmol/kg.day) Compound (mmol/kg) day (mmol/kg.day)DE-[3,4,3] 340(0.842) 87^(e)(0.215) DE-[3,4] >650^(d) (>3.22) 426(1.37)DE-[4,5,4] 285(0.638) 48(0.104) DE-[4,5] 555^(e)(1.64) 375(1.11)DE-[5,4,5] 195(0.424) 36(0.078) DE-[5,5] 500(1.42) nd All of thepolyamine analogues were administered in the form of hydrochloridesalts. ^(a)Single dose i.p. ^(b)Multiple dose i.p. (t.i.d. × 6 days)^(c)At a single dose of 250 mg/kg, no death within the initial 2 hours,but all six animals were expired within seven days. ^(d)15 mg/kg (t.i.d.× 3 days), 4/5 died on day 6 and 5/5 died on day 7. ^(e)At a single doseof 600 mg/kg, no death within the initial 2 hours, but 5/5 died withinseven days.

The study serves to define the similarities and differences betweentriamines and tetraamine analogue antineoplastics. With both tetraamineand triamine analogues, K_(i) values are sensitive to the size of theterminal substituents and the length of the backbone. This isillustrated for triamines in FIG. 8. Generally, the larger the terminalsubstituent, the more poorly the analogues are transported. In thetriamine family, spermidine analogues are the best transportcompetitors. Interestingly, the (3,3) and (5,5) triamine analogues aremost sensitive to N-terminal substituent changes. With regards touptake, the triamines are more effectively accumulated in L1210 cellsthan the corresponding tetraamines. Once in the cell, tetraamineanalogues have a greater impact on lowering overall polyamine pools;however, the triamines are more selective at reducing spermidine. Thetotal intracellular charge in picoequivalents associated withpolyamines, both native and analogues, is maintained by cells exposed toboth tetraamines and triamines. However, cells treated with triaminesare able to maintain this charge balance for a more prolonged period oftime. Both tetraamine and triamine analogues, except for DENSPD andDE(4,5), reduce ODC more effectively than AdoMetDC activity, andtetraamines are more active at this. It was demonstrated that triamineanalogue dealkylation was very specific for triamines with backbones ofless than or equal to four methylenes and most effective for triaminesand tetraamines with N^(α),N^(ω)-dipropyl substituents.

The tetraamine analogues are uniformly more active against L1210 cellsthan their triamine counterparts. With both the triamine and tetraamineanalogues the compounds' IC₅₀ values are also sensitive to the size ofthe terminal substituent and the length of the backbone. However, theoverall length between the terminal nitrogens is the most critical issuein assessing this activity; FIG. 9a illustrates the triamine case. Whencomparing N¹- with N⁸-monoalkylspermidines, the N¹ compounds, both ethyland propyl were more active than the N⁸ compound. The fact that the N¹compounds are elaborated by the cell to the corresponding and moreactive N¹-alkylspermines is in keeping with this observation. It isrecalled that the N⁸-alkylspermidines cannot be and are not furtherprocessed in the polyamine biosynthetic network. While the optimumlength for the tetraamine activity has not yet been determined (FIG.9b), evidence would suggest that the optimum length for the triamineshas been determined, as seen in the terminally dialkylated(4,5)-methylene backbone series.

The triamine analogues are less toxic than the correspondingtetraamines. Furthermore, and most important when comparing the ratio ofthe 96-hour IC₅₀/chronic LD₅₀ values of the two triamines, DE(3,4) andDE(4,5), with the corresponding tetraamines, a kind of therapeuticwindow, the triamines appear more favorable. This is a critical issue inthe choice of the best polyamine therapeutic.

The invention is illustrated by the following non-limiting exampleswherein parenthetical reference numerals correspond to those in Schemes1-5.

MENSPD (3) [Bergeron et al, Drug Metab. Dispos., Vol. 23, supra] andtetraamine analogues [Bergeron et al, J. Med. Chem., Vol. 31, supra; andBergeron et al, J. Med. Chem., Vol. 37, supra), except for DPNSPM andDE(5,4,5), were previously synthesized. N¹- and N⁸-Acetylspermidinedihydrochlorides were purchased. N-(3-aminopropyl)-1,3-propanediamine(1) was converted to its trihydrochloride salt and recrystallized fromaqueous ethanol. Sodium hydride reactions were run in distilled DMFunder an inert atmosphere. THF was distilled from sodium andbenzophenone. Fisher Optima grade solvents were routinely used, andorganic extracts were dried with sodium sulfate. Silica gel 32-63 (40 μM“flash”) was used for flash column chromatography. Melting points weredetermined on a Fisher-Johns melting point apparatus and areuncorrected. Proton NMR spectra were run at 90 or 300 MHz in CDCl₃ (notindicated) or D₂O with chemical shifts in parts per million downfieldfrom tetramethylsilane or 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid,sodium salt, respectively. Coupling constants (J) are in hertz. FAB massspectra were run in a glycerol/trifluoroacetic acid matrix. Elementalanalyses were also performed.

Cell culture materials, RPMI-1640 medium, fetal bovine serum,4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), and3-(N-morpholino)propanesulfonic acid (MOPS) were purchased. Cell numberswere determined by electronic particle analysis (Coulter Counter, ModelZF). The solid phase extraction columns (SPE-3 mL/500 mg) were used.Murine L1210 leukemia cells were obtained from the American Type TissueCorporation.

[³H]Spermidine for uptake determinations and acetyl coenzyme A(acetyl-1-¹⁴C) were purchased. L-[Carboxyl-¹⁴C]ornithine andS-adenosyl-L-[carboxyl-¹⁴C]methionine for enzyme assays were alsopurchased.

Cell Culture and IC₅₀ Determination. Murine L1210 leukemia cells (ATCCCCL 219) were maintained in logarithmic growth in RPMI-1640 mediumcontaining 10% fetal calf serum or a semisynthetic equivalent, NuSerum,2% HEPES-MOPS buffer and 1 mM aminoguanidine. The IC₅₀s, theconcentration of compound which reduces cell growth to 50% of untreatedcontrol cell growth, was determined after 48 hours and 96 hours ofexposure to polyamine analogue as detailed elsewhere [Bergeron et al, J.Med. Chem., Vol. 37, supra].

Polyamine Pool Analysis. L1210 cells in logarithmic growth were treatedwith polyamine analogue at the concentrations indicated in Table 4 for48 hours. The cells were washed twice with cold RPMI-1640, and thepellet was treated with 0.6 N HClO₄ (1 ml/10⁷ cells). Polyamine contentsof the perchloric acid extracts were quantitated by HPLC of the DANSYLderivatives [Bergeron et al, J. Med. Chem., Vol. 37, supra].

Uptake Determinations. The polyamine derivatives were studied for theirability to compete with [³H]-SPD for uptake into L1210 cells [Bergeronet al, J. Med. Chem., Vol. 37, supra]. Lineweaver-Burk plots indicated asimple competitive inhibition with respect to SPD.

Enzyme Assays. ODC and AdoMetDC activities were determined as ¹⁴CO₂released from [¹⁴C]-carboxyl-labeled L-ornithine [Seely et al,“Ornithine Decarboxylase (Mouse Kidney),” Methods Enzymol., Vol. 94,pages 158-161 (1983)] or S-adenosyl-L-methionine [Pegg et al,“S-Adenosylmethionine Decarboxylase (Rat Liver),” Methods Enzymol., Vol.94, pages 234-239 (1983)], respectively. Included in each assay wereuntreated L1210 cells as controls, as well as cells treated with DEHSPM,a drug having a known reproducible effect on each enzyme, as positivecontrols.

Spermidine/spermine N¹-acetyltransferase activity was based onquantitation of [¹⁴C]-N¹-acetylspermidine formed by acetylation of SPDwith [¹⁴C] acetyl coenzyme A according to the method of Libby et al[Biochem. Pharmacol., Vol. 38, supra]. Cells treated with DENSPM werepositive controls.

Toxicity in Mice. Acute and chronic toxicities were assessed in 10-12week old CD-1 female mice from Harlan Sprague-Dawley. For acutetoxicities, the polyamine analogues were administered in a single i.p.injection to groups of five or six animals at each dose. The animalswere scored two hours after administration of the dose. All survivorswere further observed for 10 days to assess late onset of toxicity fromthe single acute dose. In the chronic toxicity regimen, mice wereadministered polyamine analogue in three i.p. doses per day (t.i.d.) forsix days, for a total of eighteen doses per animal. Appetite, weight andoverall appearance were monitored daily. Animals were observed for 10days following the final dose, at which time the final score wasregistered. At least three test groups of 5-6 animals each, representingthree different dose levels, were evaluated for each analogue tested.These dose levels were chosen so that at least two groups presented withlethalities, one with a high fraction of lethalities (>0.50, but <1.00).

EXAMPLE 1

N¹,N⁴,N⁷-Tris(mesitylenesulfonyl)-N¹,N7-dimethylnorspermidine (43). NaH(60% in oil, 0.44 g, 11 mmol) was added to a solution of 30 [Bergeron etal, Drug Metab. Dispos., Vol. 23, supra] (3.39 g, 5 mmol) in DMF (70 mL)at 0° C. After hydrogen evolution ceased (30 minutes), iodomethane (1.63g, 11.5 mmol) was slowly added to the mixture. After stirring for 12hours at room temperature, the reaction mixture was quenched withdistilled water (10 mL). The solvents were removed under high vacuum,and the residue was combined with H₂O (30 mL) and extracted with CHCl₃(4×40 mL). The organic portion was washed with brine (80 mL) andevaporated by rotary evaporation. Purification by column chromatography(8:1 toluene/EtOAc) gave 2.47 g (70%) of 43 as an oil: NMR ε 1.73(quintet, 4 H), 2.29 (s, 6 H), 2.54-2.55 (2 s, 18 H), 2.60 (s, 6 H),2.99-3.06 (2 t, 8 H, J=7), 6.94 (s, 6 H). Anal. (C₃₅H₅₁N₃O₆S₃) C, H, N.

EXAMPLE 2

N¹,N⁷-Dimethylnorspermidine Trihydrochloride (2). HBr (30% in HOAc, 30mL) was added slowly to a mixture of 43 (2.13 g, 3.02 mmol) and phenol(12.27 g, 0.13 mol) in CH₂Cl₂ at 0° C. After stirring for 1 day at roomtemperature, H₂O (20 mL) was added to the reaction mixture followed byextraction with CH₂Cl₂ (3×30 mL). The aqueous portion was concentratedunder high vacuum, and the residue was basified to pH 14 with 1 N NaOH(4 mL) and 19 N NaOH (2 mL) and extracted with CHCl₃ (14×10 mL). Theorganic extracts were concentrated, and the residue was taken up inabsolute EtOH (40 mL) and acidified with concentrated HCl (2 mL). Afterthe solvents were removed, the solid was recrystallized from aqueousEtOH to generate 0.298 g (37%) of 2 as plates: NMR (D₂O) δ 2.08-2.19 (m,4 H), 2.75 (s, 6 H), 3.13-3.22 (m, 8 H). Anal. (C₈H₂₄Cl₃N₃) C, H, N.

EXAMPLE 3

N¹,N⁴,N⁷-Tris(mesitylenesulfonyl)-N¹,N⁷-diethylnorspermidine (44). NaH(60%, 9.0 g, 0.23 mol), 30 [Bergeron et al, Drug Metab. Dispos., Vol.23, supra] (70.0 g, 0.103 mol), and iodoethane (19 mL, 0.24 mol) in DMF(400 mL) were reacted and worked up by the method of 43. Columnchromatography (1:4 EtoAc/pet ether) afforded 63.7 g (84%) of 44 as aviscous oil: NMR δ 0.97 (t, 6 H, J=7), 1.60-1.72 (m, 4 H), 2.29 (s, 9H), 2.54 and 2.55 (2 s, 18 H), 2.95-3.15 (m, 12 H), 6.92 (s, 6 H). Anal.(C₃₇H₅₅N₃O₆S₃) C, H, N. A sample was recrystallized from EtOAc/petether, mp 97° C.

EXAMPLE 4

N¹,N⁷-Diethylnorspermidine Trihydrochloride (4). HBr (30% in HOAc, 300mL), 44 (23.0 g, 30.7 mmol), and phenol (116 g, 1.23 mol) in CH₂Cl₂ (300mL) were reacted, and product was isolated using the procedure of 2 togive 6.45 g (71%) of 4 as colorless plates: NMR (D₂O) δ 1.30 (t, 6 H,J=7), 2.05-2.18 (m, 4 H), 3.08-3.22 (m, 12 H). Anal. (C₁₀H₂₈Cl₃N₃) C, H,N.

EXAMPLE 5

N¹-Propyl-N¹,N⁴,N⁷-tris(mesitylenesulfonyl)norspermidine (45). NaH (60%,0.52 g, 13 mmol), 30 (2.57 g, 3.8 mmol), and 1-iodopropane (0.46 mL, 4.7mmol) in DMF were reacted and worked up by the method of 43. Columnchromatography (3:1 hexane/EtOAc) afforded 1.16 g (23%) of 45 as an oil:NMR δ 0.66 (t, 3 H, J=7), 1.23-1.31 (m, 2 H), 1.58-1.62 (m, 4 H),2.26-2.27 (2 s, 9 H), 2.51-2.52 (2 s, 12 H), 2.59 (s, 6 H), 2.82-2.99(m, 8 H), 3.21 (t, 2 H, J=7), 4.85 (br t, 1 H), 6.89-6.92 (m, 6 H).Anal. (C₃₆H₅₃N₃O₆S₃) C, H, N.

EXAMPLE 6

N¹-Propylnorspermidine Trihydrochloride (5). HBr (30% in HOAC, 30 mL),45 (1.14 g, 1.58 mmol), and phenol (6.4 g) in CH₂Cl₂ were reacted, andproduct was isolated using the procedure of 2 to give 87 mg (20%) of 5as crystals: NMR (D₂O) δ 0.98 (t, 3 H, J=7), 1.67-1.75 (m, 2 H),2.07-2.16 (m, 4 H), 3.01-3.21 (m, 10 H). Anal. (C₉H₂₆Cl₃N₃) C, H, N.

EXAMPLE 7

N¹,N⁷-Dipropyl-N¹,N⁴,N⁷-tris(mesitylenesulfonyl)norspermidine (46). NaH(60%, 0.44 g, 11 mmol), 30 [Bergeron et al, Drug Metab. Dispos., Vol.23, supra] (3.39 g, 5 mmol), and 1-iodopropane (1.95 g, 11.5 mmol) inDMF (70 mL) were reacted and worked up by the method of 43. Columnchromatography (3:1 hexane/EtOAc) afforded 3.54 g (93%) of 46 as an oil:NMR δ 0.7 (s, 6 H), 1.20-1.65 (m, 8 H), 2.25 (s, 9 H), 2.50 (s, 18 H),2.80-3.05 (m, 12 H), 6.87 (s, 6 H). Anal. (C₃₉H₅₉N₃O₆S₃) C, H, N.

EXAMPLE 8

N¹,N⁷-Dipropylnorspermidine Trihydrochloride (6). HBr (30% in HOAc, 80mL), 46 (3.475 g, 4.57 mmol), and phenol (15.8 g) in CH₂Cl₂ (30 mL) werereacted, and product was isolated by the procedure of 2 to provide 1.26g (85%) of 6 as plates: NMR (D₂O) δ 0.87 (t, 6 H, J=7), 1.60 (m, 4 H),2.01 (m, 4 H), 2.93 (t, 4 H, J=7), 3.06 (m, 8 H). Anal. (C₁₂H₃₂Cl₃N₃) C,H, N.

EXAMPLE 9

N¹,N⁴,N⁸-Tri(mesitylenesulfonyl)spermidine (31). Mesitylenesulfonylchloride (6.87 g, 31.4 mmol) in CH₂Cl₂ (30 mL) was added to spermidinetrihydrochloride (2.5 g, 9.8 mmol) in 1 N NaOH (35 mL) at 0° C., and themixture was efficiently stirred at room temperature overnight. Thelayers were separated, and the aqueous phase was extracted with CHCl₃(3×50 mL). The organic phase was the washed with brine (100 mL),evaporated, and purified by column chromatography (4:3 hexane/EtOAc) togive 3.73 g (55%) of 31 as a white foam: NMR δ 1.30 (m, 2 H), 1.44 (m, 2H), 1.66 (m, 2 H), 2.30 (s, 9 H), 2.46 (s, 6 H), 2.60 (s, 12 H), 2.76(q, 2 H), 2.84 (q, 2 H), 3.04 (t, 2 H, J=7), 3.24 (t, 2 H, J=7), 4.56(br t, 1 H), 4.92 (br t, 1 H), 6.90 (s, 2 H), 6.95 (s, 4 H). Anal.(C₃₄H₄₉N₃O₆S₃) C, H, N.

EXAMPLE 10

N¹,N⁸-Dimethyl-N¹,N⁴N,N⁸-tris(mesitylenesulfonyl)spermidine (47). NaH(60%, 0.41 g, 10 mmol), 31 (2.15 g, 3.11 mmol), and iodomethane (0.62mL, 10 mmol) in DMF (60 mL) were reacted and worked up as was 43. Columnchromatography (5:3 hexane/EtOAc) furnished 2.24 g (100%) of 47 as anoil: NMR δ 1.39-1.43 (m, 4 H), 1.69-1.78 (m, 2 H), 2.28 (s, 3 H), 2.30(s, 6 H), 2.55 (s, 12 H), 2.57 (s, 6 H), 2.60 (s, 3 H), 2.62 (s, 3 H),2.96-3.13 (m, 8 H).

EXAMPLE 11

N¹,N⁸-Dimethylspermidine Trihydrochloride (8). HBr (30% in HOAC, 60 mL),47 (2.24 g, 3.11 mmol), and phenol (12.3 g) in CH₂Cl₂ (30 mL) werereacted, and product was isolated by the procedure of 2 to give 0.658 g(75%) of 8 as crystals: NMR (D₂O) δ 1.77-1.82 (m, 4 H), 2.06-2.18 (m, 2H), 2.73 (s, 3 H), 2.75 (s, 3 H), 3.06-3.19 (m, 8 H). Anal. (C₉H₂₆Cl₃N₃)C, H, N.

EXAMPLE 12

N¹,N⁸-Diethyl-N¹,N⁴,N⁸-tris(mesitylenesulfonyl)spermidine (48). NaH(80%, 0.68 g, 23 mmol), 31 (7.06 g, 10.2 mmol), and iodoethane (2.5 mL,31 mmol) in DMF (75 mL) were combined as in 43. The mixture was heatedat 65° C. for 12 hours, cooled and cautiously quenched with water (70mL) and brine (100 mL), followed by extraction with EtOAc (5×100 mL).Combined organic extracts were washed with 100 mL of 1% Na₂SO₃, H₂O(2×), and brine. The solvents were removed, and the residue was purifiedby column chromatography (4.5% EtOAc/CH₂Cl₂) to produce 7.21 g (94%) of48 as an oil: NMR δ 0.8-1.8 (m, 12 H), 2.28 (s, 9 H), 2.54 (s, 18 H),2.8-3.3 (m, 12 H), 6.90 (s, 6 H). Anal. (C₃₈H₅₇N₃O₆S₃) C, H, N.

EXAMPLE 13

N¹,N⁸-Diethylspermidine Trihydrochloride (11). HBr (30% in HOAc, 150mL), 48 (7.16 g, 9.57 mmol), and phenol (28 g, 0.30 mol) in CH₂Cl₂ (125mL) were reacted, and product was isolated utilizing the procedure of 2to give 2.17 g (73%) of 11 as white plates: NMR (D₂O) δ 1.28 and 1.30 (2t, 6 H, J=7), 1.73-1.85 (m, 4 H), 2.06-2.18 (m, 2 H), 3.04-3.21 (m, 12H). Anal. (C₁₁H₃₀Cl₃N₃) C, H, N.

EXAMPLE 14

N¹,N⁸-Dipropyl-N¹,N⁴,N⁸-tris(mesitylenesulfonyl)spermidine (49). NaH(80%, 0.80 g, 27 mmol) was added to 31 (8.13 g, 11.7 mmol) in DMF (75mL) at 0° C. The mixture was stirred at room temperature for 1 hour, and1-iodopropane (3.5 mL, 36 mmol) was added by syringe. The mixture wasstirred at 80° C. for 12 hours and worked up as was 48. Purification bycolumn chromatography (3.5% EtOAc/CH₂Cl₂) resulted in 8.42 g (93%) of 49as an oil: NMR δ 0.55-1.72 (m, 16 H), 2.25 (s, 9 H), 2.50 (s, 18 H),2.7-3.3 (m, 12 H), 6.87 (s, 6 H). Anal. (C₄₀H₆₁N₃O₆S₃) C, H, N.

EXAMPLE 15

N¹,N⁸-Dipropylspermidine Trihydrochloride (14). HBr (30% in HOAc, 150mL), 49 (8.32 g, 10.7 mmol), and phenol (28 g, 0.29 mol) in CH₂Cl₂ (125mL) were reacted, and product was isolated by the procedure of 2 toproduce 2.58 g (71%) of 14 as white plates: NMR (D₂O) δ 0.97 and 0.98 (2t, 6 H, J=7), 1.63-1.83 (m, 8 H), 2.06-2.19 (m, 2 H), 2.97-3.21 (m, 12H). Anal. (C₁₃H₃₄Cl₃N₃) C, H, N.

EXAMPLE 16

N,N′-Bis(4-Phthalimidobutyl)mesitylenesulfonamide (37). NaH (60%, 1.6 g,40 mmol) was added to 35 [Schreinemakers, Recl. Trav. Chim. Pays-BasBelg., Vol. 16, supra] (2.72 g, 13.5 mmol) in DMF (60 mL) at 0° C. Afterthe mixture was stirred at 0° C. for 30 minutes,N-(4-bromobutyl)phthalimide (11.51 g, 40 mmol) in DMF (20 mL) wasintroduced. The mixture was stirred at room temperature for 1 hour andat 60° C. overnight. Following the workup procedure of 43, columnchromatography (25:1 CHCl₃/acetone) gave 3.77 g (46%) of 37 as a whitepowder: NMR δ 1.51-1.54 (m, 8 H), 2.18 (s, 3 H), 2.57 (s, 6 H),3.18-3.24 (m, 4 H), 3.55-3.60 (m, 4 H), 6.86 (s, 2 H), 7.69-7.25 (m, 4H), 7.82-7.85 (m, 4 H). HRMS calcd. for C₃₃H₃₆N₃O₆S 602.2325 (M+H),found. 602.2320 (M+H).

EXAMPLE 17

N,N′-Bis(4-aminobutyl)mesitylenesulfonamide (40). Hydrazine monohydrate(0.82 g, 16 mmol) was added to a suspension of 37 (3.5 g, 5.8 mmol) inabsolute EtOH (100 mL), and the mixture was stirred at 65° C. for 24hours. After cooling, the solid was filtered and washed with EtOH (2×10mL). The combined filtrate was concentrated and purified by columnchromatography (6:1 MeOH/concentrated NH₄OH) to produce 1.50 g (76%) of40 as a viscous oil: NMR δ 1.37 (quintet, 4 H), 1.52 (quintet, 4 H),2.30 (s, 3 H), 2.54 (t, 4 H, J=7), 2.58 (s, 6 H), 3.20 (t, 4 H, J=7),7.04 (s, 3 H).

EXAMPLE 18

Homospermidine Trihydrochloride (15). HBr (30% in HOAC, 30 mL), 40 (1.50g, 4.39 mmol) and phenol (4.49 g, 48 mmol) in CH₂Cl₂ (20 mL) werereacted, and product was isolated by the procedure of 2 to afford 0.86 g(73%) of 15 as white crystals: NMR (D₂O) δ 1.73-1.80 (m, 8 H), 3.03-3.14(m, 8 H). Anal. (C₈H₂₄Cl₃N₃) C, H, N.

EXAMPLE 19

N¹,N⁵,N⁹-Tris(mesitylenesulfonyl)homospermidine (32). Mesitylenesulfonylchloride (6.71 g, 30.7 mmol) and 40 (4.76 g, 14 mmol) in CH₂Cl₂ (30 mL)and 1 N NaOH (35 mL) were combined and worked up by the method of 31.Column chromatography (4:1 toluene/EtOAc) produced 3.06 g (31%) of 32 asa white foam: NMR δ 1.32-1.38 (m, 4 H), 1.44-1.54 (m, 4 H), 2.28-2.29 (2s, 9 H), 2.54 (s, 6 H), 2.60 (s, 12 H), 2.79 (quartet, 4 H), 3.09 (t, 4H, J=7), 4.70-4.80 (br s, 2 H), 6.90 (s, 2 H), 6.92 (s, 4 H). Anal.(C₃₅H₅₁N₃O₆S₃) C, H, N.

EXAMPLE 20

N¹,N⁹-Dimethyl-N¹,N⁵,N⁹-tris(mesitylenesulfonyl)homospermidine (50). NaH(60%, 0.17 g, 4.2 mmol), 32 (1.28 g, 1.8 mmol), and iodomethane (0.25mL, 4.0 mmol) in DMF (50 mL) were reacted and worked up as was 43.Column chromatography (2:1 toluene/EtOAc) gave 1.14 g (86%) of 50 as anoil: NMR δ 1.38-1.44 (m, 8 H), 2.28 (s, 3 H), 2.30 (s, 6 H), 2.57 (s, 18H), 2.62 (s, 6 H), 3.03-3.14 (m, 8 H), 6.93-6.94 (2 s, 6 H). Anal.(C₃₇H₅₅N₃O₆S₃) C, H, N.

EXAMPLE 21

N¹,N⁹-Dimethylhomospermidine Trihydrochloride (16). HBr (30% in HOAC, 30mL), 50 (1.12 g, 1.52 mmol), and phenol (5.4 g, 57 mmol) in CH₂Cl₂ (25mL) were reacted, and product was isolated by the procedure of 2 toprovide 354 mg (79%) of 16 as plates: NMR (D₂O) δ 1.78 (m, 8 H), 2.73(s, 6 H), 3.08-3.12 (m, 8 H). Anal. (C₁₀H₂₈Cl₃N₃) C, H, N.

EXAMPLE 22

N¹,N⁹-Diethyl-N¹,N⁵,N⁹-tris(mesitylenesulfonyl)homospermidine (51). NaH(80%, 0.264 g, 8.8 mmol) was added to 35 [Schreinemakers, Recl. Trav.Chim. Pays-Bas Belg., Vol. 16, supra] (0.796 g, 4 mmol) in DMF (60 mL)at 0° C. After the mixture was stirred at 0° C. for 30 minutes, 58[Bergeron et al, J. Med. Chem., Vol. 37, supra]. (3.19 g, 8.8 mmol) inDMF (15 mL) was added. The mixture was heated at 75° C. overnight andworked up by the procedure of 43. Column chromatography (3:1hexane/EtOAc) gave 2.82 g (93%) of 51 as an oil: NMR δ 0.96 (t, 6 H),1.20-1.40 (m, 8 H), 2.25 (s, 9 H), 2.55 (s, 18 H), 2.85-3.20 (m, 12 H),6.90 (s, 6 H). Anal. (C₃₉H₅₉N₃O₆S₃) C, H, N.

EXAMPLE 23

N¹,N⁹-Diethylhomospermidine Trihydrochloride (17). HBr (30% in HOAC, 20mL), 51 (1.87 g, 2.45 mmol), and phenol (4.4 g, 49 mmol) in CH₂Cl₂ (20mL) were reacted, and product was isolated by the procedure of 2 to give493 mg (62%) of 17 as plates: NMR (D₂O) δ 1.30 (s, 6 H), 1.55-1.90 (m, 8H), 2.95-3.20 (m, 12 H). Anal. (C₄₁H₆₃N₃O₃N₃) C, H, N.

EXAMPLE 24

N¹,N⁹-Dipropyl-N¹,N⁵,N⁹-tris(mesitylenesulfonyl)homospermidine (52). NaH(60%, 0.17 g, 4.2 mmol), 32 (1.28 g, 1.8 mmol), and 1-iodopropane (0.39mL, 4.0 mmol) in DMF (50 mL) were reacted and worked up using theprocedure of 43. Column chromatography (4:1 hexane/EtOAc) gave 1.26 g(89%) of 52 as an oil: NMR δ 0.74 (t, 6 H, J=7), 1.26-1.45 (m, 12 H),2.29 (s, 9 H), 2.55 (s, 18 H), 2.98-3.13 (m, 12 H), 6.87 (s, 6 H). Anal.(C₄₁H₆₃N₃O₆S₃) C, H, N.

EXAMPLE 25

N¹,N⁹-Dipropylhomospermidine Trihydrochloride (19). HBr (30% in HOAc, 30mL), 52 (1.24 g, 1.56 mmol), and phenol (5.4 g, 57 mmol) in CH₂Cl₂ (25mL) were reacted, and product was isolated by the procedure of 2 to give430 mg (78%) of 19 as plates: NMR (D₂O) δ 0.98 (t, 6 H, J=7), 1.70 (m, 4H), 1.76-1.80 (m, 8H), 3.02 (t, 4 H, J=7), 3.08-3.12 (m, 8 H). Anal.(C₁₄H₃₆Cl₃N₃) C, H, N.

EXAMPLE 26

N-(3-Cyanopropyl)mesitylenesulfonamide (36). NaH (60%, 2.0 g, 50 mmol),35 [Schreinemakers, Recl. Trav. Chim. Pays-Bas Belg., Vol. 16, supra](10.0 g, 50 mmol), and 4-bromobutyronitrile (4 mL, 40 mol) in DMF (100mL) were combined. The mixture was heated at 80° C. overnight and workedup by the procedure of 43. Column chromatography (4:3 hexane/EtOAc) gave5.04 g (38%) of 36 as an oil: NMR δ 1.78 (s, 3 H), 2.25 (s, 3 H), 2.35(t, 2 H, J=7), 2.95 (q, 2 H), 5.05 (br t, 1 H), 6.90 (s, 2 H). Anal.(C₁₃H₁₈N₂O₂S) C, H, N.

EXAMPLE 27

N-(4-Cyanobutyl)-N-(3-cyanopropyl)mesitylenesulfonamide (38). NaH (60%,0.90 g, 23 mmol), 36 (5.02 g, 18.85 mmol), and 5-bromovaleronitrile (2.4mL, 21 mmol) in DMF were combined and worked up by the method of 43.Column chromatography (1:1 hexane/EtOAc) provided 5.20 g (79%) of 38 asan oil: NMR δ 1.49-1.66 (m, 4 H), 1.82 (m, 2 H), 2.22 (t, 2 H, J=7),2.25 (t, 2 H, J=7), 2.29 (s, 3 H), 2.57 (s, 6 H), 3.19 (t, 2 H, J=7),3.29 (t, 2 H, J=7), 6.95 (s, 2 H). Anal. (C₁₈H₂₅N₃O₂S) C, H, N.

EXAMPLE 28

6-(Mesitylenesulfonyl)-1,6,12-Triazadodecane (41). Raney nickel (W-2grade, 7.60 g) and concentrated NH₄OH (10 mL) were successively added to38 (5.06 g, 14.6 mmol) in CH₃OH (30 mL) and THF (30 mL) in a 200 mL Parrbottle, and a slow stream of NH₃ was bubbled through the mixture for 30minutes at 0° C. After hydrogenation in a Parr bottle was carried out at50-55 psi for 8 hours, the suspension was filtered through Celite, andthe solvents were removed in vacuo to give 4.70 g (91%) of 41 as an oil:NMR δ 1.14-1.24 (m, 10 H), 2.25 (s, 3 H), 2.6 (s, 6 H), 3.05-3.25 (m, 8H), 3.45 (s, 4 H), 6.9 (s, 2 H).

EXAMPLE 29

1,6,12-Triazadodecane Trihydrochloride (20). HBr (30% in HOAC, 33 mL),41 (2.43 g, 6.83 mmol), and phenol (6 g, 60 mmol) in CH₂Cl₂ werereacted, and product was isolated by the procedure of 2 to give 0.97 g(50%) of 20 as a hygroscopic solid: NMR (D₂O) δ 1.47 (m, 2 H), 1.70-1.80(m, 8 H), 3.00-3.10 (m, 8 H). Anal. (C₉H₂₆Cl₃N₃) C, H, N.

EXAMPLE 30

1,6,12-Tris(mesitylenesulfonyl)-1,6,12-Triazadodecane (33).Mesitylenesulfonyl chloride (4.29 g, 19.6 mmol) and 41 (3.17 g, 8.92mmol) in CH₂Cl₂ (40 mL) and 1 N NaOH (20 mL) were combined and worked upby the method of 31. Column chromatography (4:3 hexane/EtOAc) generated5.66 g (88%) of 33 as an oil: NMR δ 1.12-1.17 (m, 2 H), 1.34-1.51 (m, 8H), 2.29 (s, 3 H), 2.30 (s, 6 H), 2.55 (s, 6 H), 2.60-2.62 (2 s, 12 H),2.77-2.81 (m, 4 H), 3.06 (t, 2 H, J=7), 3.11 (t, 2 H, J=7), 4.50-4.60(m, 2 H), 6.92 (s, 2 H), 6.95 (s, 2 H). Anal. (C₃₆H₅₃N₃O₆S₃) C, H, N.

EXAMPLE 31

2,7,13-Tris(mesitylenesulfonyl)-2,7,13-Triazapentadecane (53). NaH (60%,0.28 g, 6.9 mmol), 33 (2.16 g, 3.0 mmol), and iodomethane (6.1 mL, 9.8mmol) in DMF (30 mL) were combined and worked up by the method of 43.Column chromatography (7:3 hexane/EtOAc) gave 1.90 g (85%) of 53 as anoil: NMR δ 1.08-1.16 (m, 2 H), 1.38-1.50 (m, 8 H ), 2.28-2.29 (2 s, 9H), 2.57-2.58 (2 s, 18 H), 2.63 (s, 3 H), 2.65 (s, 3 H), 3.02-3.14 (m, 8H), 6.95 (s, 6 H); HRMS calcd. for C₃₈H₅₈N₃O₆S₃ 748.3487 (M+H), found.748.3483 (M+H).

EXAMPLE 32

2,7,13-Triazatetradecane Trihydrochloride (21). HBr (30% in HOAC, 45mL), 53 (1.85 g, 2.47 mmol), and phenol (8.5 g) in CHCl₂ (20 mL) werereacted, and product was isolated by the procedure of 2 to give 529 mg(69%) of 21 as crystals: NMR (D₂O) δ 1.42-1.52 (m, 2 H), 1.69-1.81 (m, 8H), 2.73-2.74 (2 s, 6 H), 3.03-3.12 (m, 8 H). Anal. (C₁₁H₃₀Cl₃N₃) C, H,N.

EXAMPLE 33

4,9,15-Tris(mesitylenesulfonyl)-4,9,15-Triazaheptadecane (54). NaH (60%,0.273 g, 6.84 mmol), 33 (2.24 g, 3.11 mmol), and 1-iodopropane (0.67 mL,6.9 mmol) in DMF (30 mL) were combined and worked up by the method of43. Column chromatography (3:1 hexane/EtOAc) provided 2.01 g (80%) of 54as an oil: NMR δ 0.71-0.78 (m, 6 H), 1.01-1.11 (m, 2 H), 1.34-1.48 (m,12 H), 2.29 (s, 6 H), 2.57-2.58 (2 s, 18 H), 2.98-3.13 (m, 12 H), 6.92(s, 6 H). Anal. (C₄₂H₆₅N₃O₆S₃.H₂O) C, H, N.

EXAMPLE 34

4,9,15-Triazaoctadecane Trihydrochloride (23). HBr (30% in HOAC, 45 mL),48 (1.99 g, 2.47 mmol), and phenol (8.5 g) in CH₂Cl₂ (20 mL) werereacted, and product was isolated by the procedure of 2 to give 852 mg(83%) of 23 as plates: NMR (D₂O) δ 0.97 (s, 6 H), 1.40-1.51 (m, 2 H),1.66-1.80 (m, 12 H), 2.98-3.15 (m, 12 H). Anal. (C₁₅H₃₈Cl₃N₃) C, H, N.

EXAMPLE 35

N,N-Bis(4-cyanobutyl)mesitylenesulfonamide (39). NaH (80%, 1.22 g, 51mmol), 35 [Schreinemakers, Recl. Trav. Chim. Pays-Bas Belg., Vol. 16,supra) (5.0 g, 25 mmol), and 5-chlorovaleronitrile (6.5 g, 55 mmol) inDMF (50 mL) were combined. The mixture was heated at 60° C. overnightand worked up by the procedure of 43. Column chromatography (7:3hexane/EtOAc) yielded 6.31 g (70%) of 39 as an oil: NMR δ 1.57 (m, 4 H),1.66 (m, 4 H), 2.26 (t, 4 H, J=7), 2.60 (s, 6 H), 3.22 (t, 4 H, J=7),6.98 (s, 2 H). Anal. (C₁₉H₂₇N₃O₂S) C, H, N.

EXAMPLE 36

7-Mesitylenesulfonyl-1,7,13-triazatridecane (42). Raney nickel (W-2grade, 2.9 g) and 39 (5.69 g, 15.8 mmol) in concentrated NH₄OH (10 mL)and CH₃OH (60 mL) were saturated with NH₃ as 41. The mixture was shakenwith hydrogen at 50-55 psi in a 200 mL Parr bottle for 42 hours. Thesuspension was filtered through Celite, and the solvents were removed invacuo. The residue was passed through a short silica gel column (EtOHthen 5% concentrated NH₄OH/EtOH) to give 5.71 g (98%) of 42 as a lightyellow oil: NMR δ 1.17 (m, 4 H), 1.47 (m, 8 H), 2.27 (s, 3 H), 2.57 (s,6 H), 2.61 (m, 4 H), 3.13 (t, J=7.5, 4 H), 6.91 (s, 2 H); HRMS calcd.for C₁₉H₃₆N₃O₂S 370.2528 (M+H), found. 370.2530 (M+H).

EXAMPLE 37

1,7,13-Triazatridecane Trihydrochloride (24). HBr (30% in HOAc, 26 mL),42 (2.0 g, 5.42 mmol), and phenol (4.8 g, 51 mmol) in CHCl₃ (40 mL) werereacted, and product was isolated by the method of 2 to give 0.97 (61%)of 24 as a white solid: NMR (D₂O) δ 1.45 (m, 4 H), 1.70 (m, 8 H), 3.01(m, 8 H). Anal. (C₁₀H₂₈Cl₃N₃) C, H, N.

EXAMPLE 38

1,7,13-Tris(mesitylenesulfonyl)-1,7,13-triazatridecane (34).Mesitylenesulfonyl chloride (4.52 g, 20.7 mmol) and 42 (3.47 g, 9.4mmol) in CH₂Cl₂ and 1 N NaOH (30 mL) were combined and worked up by themethod of 31. Column chromatography (3:2 hexane/EtOAc) gave 6.44 g (93%)of 34 as a white solid: NMR δ 1.16 (m, 4 H), 1.39 (m, 8 H), 2.30 (s, 3H), 2.31 (s, 6 H), 2.57 (s, 6 H), 2.62 (s, 12 H), 2.81 (d of t, 4 H),3.10 (t, 4 H, J=7), 4.49 (br t, 2 H), 6.95 (s, 2 H), 6.97 (s, 4 H); HRMScalcd. for C₃₇H₅₆N₃O₆S₃ 734.3331 (M+H), found. 734.3351 (M+H).

EXAMPLE 39

2,8,14-Tris(mesitylenesulfonyl)-2,8,14-triazapentadecane (55). NaH (80%,0.207 g, 6.9 mmol), 34 (1.58 g, 2.16 mmol), and iodomethane (0.30 mL,4.8 mmol) in DMF (30 mL) were reacted and worked up as was 43. Columnchromatography (5:2 hexane/EtOAc) gave 1.51 g (92%) of 55 as an oil: NMRδ 1.06-1.18 (m, 4 H), 1.40-1.52 (m, 8 H), 2.29 (s, 9 H), 2.59 (s, 18 H),2.66 (s, 6 H), 3.03-3.14 (m, 8 H), 6.95 (s, 6 H). Anal. (C₃₉H₅₉N₃O₆S₃)C, H, N.

EXAMPLE 40

2,8,14-Triazapentadecane Trihydrochloride (25). HBr (30% in HOAc, 30mL), 55 (1.48 g, 1.94 mmol), and phenol (5.2 g, 55 mmol) in CH₂Cl₂ (30mL) were reacted, and product was isolated by the method of 2 to produce480 mg (76%) of 25 as needles: NMR (D₂O) δ 1.4-1.5 (quintet, 4 H),1.7-1.8 (quintet, 8 H), 2.7 (s, 6 H), 3.05 (t, 8 H, J=7). Anal.(C₁₂H₃₂Cl₃N₃) C, H, N.

EXAMPLE 41

3,9,15-Tris(mesitylenesulfonyl)-3,9,15-triazaheptadecane (56). NaH (80%,0.52 g, 17 mmol), 34 (3.2 g, 4.36 mmol), and iodoethane (1.5 g, 9.6mmol) in DMF (20 mL) were reacted and worked up by the method of 43.Column chromatography (4:1 hexane/EtOAc) gave 2.91 g (85%) of 56 as awhite solid: mp 60-62° C.; NMR δ 1.01 (t, 6 H, J=7), 1.08 (m, 4 H), 1.42(m, 8 H), 2.29 (s, 9 H), 2.57 (s, 6 H), 2.58 (s, 12 H), 3.07 (t, 4 H,J=7), 3.11 (t, 4 H, J=7), 3.17 (q, 4 H), 6.92 (s, 6 H). Anal.(C₄₁H₆₃N₃O₆S₃) C, H, N.

EXAMPLE 42

3,9,15-Triazaheptadecane Trihydrochloride (26). HBr (30% in HOAc, 20mL), 56 (2.9 g, 3.67 mmol), and phenol (3.25 g, 34.5 mmol) in CHCl₃ (27mL) were reacted, and product was isolated by the method of 2 to give1.0 g (77%) of 26 as white crystals: NMR (D₂O) δ 1.28 (t, 6 H, J=7),1.45 (m, 4 H), 1.73 (m, 8 H), 3.08 (m, 12 H). Anal. (C₁₄H₃₆Cl₃N₃) C, H,N.

EXAMPLE 43

4,10,16-Tris(mesitylenesulfonyl)-4,10,16-triazanonadecane (57). NaH(80%, 198 mg, 6.6 mmol), 34 (1.51 g, 2.06 mmol), and 1-iodopropane (0.44mL, 4.5 mmol) in DMF (40 mL) were reacted and worked up by the method of43. Column chromatography (7:2/hexane/EtOAc) provided 1.61 g (95%) of 57as an oil: NMR δ 0.75 (t, 6 H, J=7), 1.02-1.14 (m, 4 H), 1.36-1.52 (m,12 H), 2.30 (s, 9 H), 2.60 (s, 18 H), 3.02-3.16 (m, 12 H), 6.95 (s, 6H). Anal. (C₄₃H₆₇N₃O₆S₃) C, H, N.

EXAMPLE 44

4,10,16-Triazanonadecane Trihydrochloride (27). HBr (30% in HOAC, 30mL), 57 (1.58 g, 1.93 mmol), and phenol (5.2 g, 55 mmol) were reacted,and product was isolated by the method of 2 to give 579 mg (79%) of 27as plates: NMR (D₂O) δ 0.95 (t, 6 H, J=7), 1.38-1.49 (m, 4 H), 1.62-1.77(m, 12 H), 2.95-3.05 (m, 12 H). Anal. (C₁₆H₄₀Cl₃N₃) C, H, N.

EXAMPLE 45

N-(5-Bromopentyl)-N-ethylmesitylenesulfonamide (61). NaH (80%, 1.26 g,42 mmol), 60 [Schreinemakers, Recl. Trav. Chim. Pays-Bas Belg., Vol. 16,supra] (6.82 g, 30.0 mmol), and 1,5-dibromopentane (49 mL, 0.36 mol) inDMF (100 mL) were combined. The mixture was heated at 74° C. overnightand worked up by the procedure of 43. Column chromatography (7:1hexane/EtOAc) produced 7.87 g (70%) of 61 as an oil: NMR δ 1.00 (t, 3 H,J=7), 1.30-1.75 (m, 6 H), 2.20 (s, 3 H), 2.50 (s, 6 H), 3.02-3.30 (m, 6H), 6.80 (s, 2 H); HRMS calcd. for C₁₆H₂₇BrNO₂S 376.0946 (M+H), found.376.0960 (M+H).

EXAMPLE 46

N¹,N⁴-Bis(mesitylenesulfonyl)-N¹-(tert-butoxycarbonyl)-N⁴-ethyl-1,4-diaminobutane(62). NaH (80%, 0.45 g, 23 mmol) was added to 59 (Bergeron et al, J.Med. Chem., Vol. 37, supra]. (3.45 g, 11.5 mmol) in DMF (100 mL) at 0°C. After the mixture was stirred at 0° C. for 40 minutes, 58 [Bergeronet al, J. Med. Chem., Vol. 37, supra]. (5.00 g, 13.8 mmol) in DMF (10mL) was added. The mixture was heated at 60° C. for 18 hours and thenworked up by the method of 43. Column chromatography with (20:1toluene/EtOAc) gave 6.44 g (96%) of 62 as an oil: NMR δ 1.12 (t, 3 H,J=7), 1.20 (s, 9 H), 1.55-1.65 (m, 4 H), 2.29 (s, 2 H), 2.30 (s, 2 H),2.52 (s, 4 H), 2.62 (s, 4 H), 3.16-3.24 (m, 2 H), 3.30 (q, 2 H), 3.70(m, 2 H), 6.94 (s, 4 H).

EXAMPLE 47

N¹,N⁴-Bis(mesitylenesulfonyl)-N-ethyl-1,4-diaminobutane (63). TFA (70mL) was slowly dripped into a solution of 62 (6.20 g, 10.6 mmol) inCH₂Cl₂ (30 mL) at 0° C. After the solution was stirred at 0° C. for 20minutes and at room temperature for 30 minutes, solvents were removed byrotary evaporation. The residue was basified to pH >8 with saturatedNaHCO₃ and extracted with CH₂Cl₂ (4×100 mL). Removal of organic extractsled to 5.10 g (100%) of 63 as a foam: NMR δ 0.97 (t, 3 H, J=7),1.20-1.50 (m, 4 H), 2.25 (s, 6 H), 2.50-2.55 (2 s, 12 H), 2.95-3.25 (m,6 H), 4.45 (t, 1 H), 6.85 (s, 4 H). Anal. (C₂₄H₃₆N₂O₄S₂) C, H, N.

EXAMPLE 48

3,8,14-Tris(mesitylenesulfonyl)-3,8,14-Triazahexadecane (64). NaH (80%,0.41 g, 14 mmol) was added to 63 (5.10 g, 10.6 mmol) in DMF (50 mL) at0° C. After the mixture was stirred at 0° C. for 30 minutes, 61 (4.80 g,12.7 mmol) in DMF (10 mL) was added. The mixture was heated at 89° C.overnight and then worked up by the method of 43. Column chromatography(12:1 toluene/EtOAc) gave 5.43 g (66%) of 64 as an oil: NMR δ 0.9-1.1(m, 6 H), 1.2-1.5 (m, 10 H), 2.25 (s, 6 H), 2.30 (s, 3 H), 2.55 (s, 18H), 2.9-3.2 (m, 12 H), 6.85 (s, 6 H). Anal. (C₄₀H₆₁N₃O6S₃) C, H, N.

EXAMPLE 49

3,8,14-Triazahexadecane Trihydrochloride (22). HBr (30% in HOAc, 100mL), 64 (5.4 g, 7.0 mmol), and phenol (25 g, 0.28 mol) were reacted, andproduct was isolated by the method of 2 to give 1.63 g (69%) of 22 asplates: NMR (D₂O) δ 1.38 (t, 3 H, J=7), 1.39 (t, 3 H, J=7), 1.60-1.70(m, 10 H), 3.02-3.15 (m, 12 H). Anal. (C₁₃H₃₄Cl₃N₃) C, H, N.

EXAMPLE 50

N¹-Triphenylmethyl-1,3-diaminopropane (68). A solution oftriphenylmethyl chloride (6.97 g, 25 mmol) in CH₂Cl₂ (100 mL) was addeddropwise to a rapidly stirred solution of 1,3-diaminopropane (9.86 g,133 mmol) in CH₂Cl₂ (100 mL). After stirring at room temperature for 2days, 1 N NaOH (50 mL) was added to the mixture, which was extractedwith CHCl₃ (3×50 mL). Organic extracts were washed with 100 mL of H₂Oand brine. After solvent removal, column chromatography (3% concentratedNH₄OH/MeOH) gave 6.32 g (80%) of 68 as a white solid: mp 59-61° C. [Parget al, “A Semiconducting Langmuir-Blodgett Film of a Non-amphiphilicBis-tetrathiofulvalene Derivative,” J. Mater. Chem., Vol. 5, pages1609-1615 (1995); mp 59-61° C.]; NMR δ 1.35-1.65 (m, 6 H), 2.18 (t, 2 H,J=7), 2.77 (s, 1 H), 7.13-7.24 (m, 9 H), 7.44-7.46 (m, 6 H). Anal.(C₂₂H₂₄N₂) C, H, N.

EXAMPLE 51

N¹-Mesitylenesulfonyl-N³-triphenylmethyl-1,3-diaminopropane (70).Mesitylenesulfonyl chloride (5.25 g, 24 mmol) and 68 (6.30 g, 20 mmol)in CH₂Cl₂ (30 mL) and 1 N NaOH (27 mL) were combined and worked up bythe method of 31. Column chromatography (7:2 hexane/EtOAc) gave 8.37 g(84%) of 70 as a white solid: NMR δ 1.56-1.66 (m, 3 H), 2.17 (t, 2 H,J=7), 2.29 (s, 3 H), 2.60 (s, 6 H), 3.08 (q, 2 H), 5.25 (br t, 1 H),6.93 (s, 2 H), 7.15-7.28 (m, 9 H), 7.35-7.39 (m, 6 H). Anal.(C₃₁H₃₄N₂O₂S) C, H, N.

EXAMPLE 52

N¹-Triphenylmethyl-1,4-diaminobutane (69). A solution of triphenylmethylchloride (59.94 g, 0.215) in CH₂Cl₂ (500 mL) was added dropwise to arapidly stirred solution of 1,4-diaminobutane (96.47 g, 1.094 mol) inCH₂Cl₂ (1.1 L) over a period of 2 hours. The reaction mixture wasstirred at room temperature for 3 days and was worked up following themethod of 68 to give a quantitative yield of 69 as an oil which was useddirectly in the next step: NMR δ 1.39-1.54 (m, 7 H), 2.12 (t, 2 H, J=7),2.62 (t, 2 H, J=7), 7.13-7.28 (m, 9 H), 7.41-7.47 (m, 6 H).

EXAMPLE 53

N¹-Mesitylenesulfonyl-N⁴-triphenylmethyl-1,4-diaminobutane (71).Mesitylenesulfonyl chloride (3.1 g, 14 mmol) and 69 (3.39 g, 10.3 mmol)in CH₂Cl₂ (20 mL) and 1 N NaOH (15 mL) were combined and worked up bythe method of 31. Column chromatography (1:3 hexane/EtOAc) furnished3.79 g (72%) of 71 as a white solid: NMR δ 1.41-1.50 (m, 5 H), 2.03-2.08(t, 2 H, J=7), 2.27 (s, 3 H), 2.62 (s, 6 H), 2.87 (q, 2 H), 4.41 (br t,1 H), 6.93 (s, 2 H), 7.15-7.28 (m, 9 H), 7.40-7.44 (m, 6 H). Anal.(C₃₂H₃₆N₂O₂S) C, H, N.

EXAMPLE 54

N-Propylmesitylenesulfonamide (65). Mesitylenesulfonyl chloride (12.0 g,55 mmol) and propylamine (2.96 g, 50 mmol) in CH₂Cl₂ (60 mL) and 1 NNaOH (60 mL) were combined and worked up by the method of 31. Columnchromatography (3:1 hexane/EtOAc) afforded 8.44 g (85%) of 65 as acrystalline solid: mp 53-54° C.; NMR δ 0.86 (t, 3 H, J=7), 1.44-1.51 (m,2 H), 2.30 (s, 3 H), 2.64 (s, 6 H), 2.86 (q, 2 H), 4.40 (br t, 1 H),6.96 (s, 2 H). Anal. (C₁₂H₁₉NO₂S) C, H, N.

EXAMPLE 55

N-(3-Bromopropyl)-N-propylmesitylenesulfonamide (66). NaH (60%, 0.34 g,8.4 mmol), 65 (1.7 g, 7.0 mmol), and 1,3-dibromopropane (17.0 g, 84mmol) in DMF (30 mL) were combined and worked up by the method of 43.Column chromatography (6:1 hexane/EtOAc) produced 1.82 g (80%) of 66 asan oil: NMR δ 0.79 (s, 3 H), 1.48-1.56 (m, 2 H), 2.04-2.10 (m, 2 H),2.30 (s, 3 H), 2.60 (s, 6 H), 3.09-3.14 (m, 2 H), 3.29-3.36 (m, 4 H).Anal. (C₁₅H₂₄BrNO₂S) C, H, N.

EXAMPLE 56

N-(4-Bromobutyl)-N-propylmesitylenesulfonamide (67). NaH (60%, 0.70 g,17 mmol), 65 (3.5 g, 14.5 mmol), and 1,4-dibromobutane (37.6 g, 174mmol) in DMF (40 mL) were combined and worked up by the method of 43.Excess 1,4-dibromobutane was removed by a Kugelrohr apparatus under highvacuum. Column chromatography (7:1 hexane/EtOAc) produced 5.21 g (95%)of 67 as an oil: NMR δ 0.79 (t, 3 H, J=7), 1.46-1.54 (m, 2 H), 1.64-1.78(m, 4 H), 2.30 (s, 3 H), 2.60 (s, 6 H), 3.11 (t, 2 H, J=7), 3.21 (t, 2H, J=7), 3.31 (t, 2 H, J=7), 6.93 (s, 2 H). Anal. (C₁₆H₂₆BrNO₂S) C, H,N.

EXAMPLE 57

6,10-Bis(mesitylenesulfonyl)-1-triphenylmethyl-1,6,10-triazatridecane(72). NaH (60%, 0.24 g, 5.96 mmol) was added to 71 (2.55 g, 4.97 mmol)in DMF (40 mL) at 0° C. After the mixture was stirred at 0° C. for 20minutes, 66 (1.80 g, 4.97 mmol) in DMF (20 mL) was added. The mixturewas stirred at room temperature for 1 day and then worked up followingthe method of 43. Column chromatography (20:1 toluene/EtOAc) produced3.72 g (94%) of 72 as an oil: NMR δ 0.73 (t, 3 H, J=7), 1.26-1.70 (m, 8H), 2.01 (t, 2 H, J=7), 2.26 (s, 3 H), 2.27 (s, 3 H), 2.54 (s, 12 H),2.96-3.04 (m, 8 H), 6.90 (s, 4 H), 7.18-7.45 (m, 15 H). Anal.(C₄₇H₅₉N₃O₄S₂) C, H, N.

EXAMPLE 58

5,10-Bis(mesitylenesulfonyl)-1-triphenylmethyl-1,5,10-triazatridecane(73). NaH (60%, 0.22 g, 5.42 mmol), 70 (2.25 g, 4.52 mmol) in DMF (40mL), and 67 (1.70 g, 4.52 mmol) in DMF (20 mL) were reacted and workedup following the method of 72. Column chromatography (25:1toluene/EtOAc) produced 2.87 g (80%) of 73 as an oil: NMR δ 0.75 (t, 3H, J=7), 1.41-1.46 (m, 9 H), 1.97 (t, 2 H, J=7), 2.26 (s, 6 H), 2.53 (s,6 H), 2.56 (s, 6 H), 3.04 (t, 2 H, J=7), 3.10-3.15 (m, 6 H), 6.88 (s, 2H), 6.90 (s, 2 H), 7.15-7.38 (m, 15 H). Anal. (C₄₇H₅₉N₃O₄S₂) C, H, N.

EXAMPLE 59

6,11-Bis(mesitylenesulfonyl)-1-triphenylmethyl-1,6,11-triazatetradecane(74). NaH (60%, 0.12 g, 2.90 mmol), 71 (1.24 g, 2.42 mmol) in DMF (30mL), and 67 (0.91 g, 2.42 mmol) in DMF (10 mL) were reacted and workedup following the method of 72. Column chromatography (25:1toluene/EtOAc) gave 1.67 g (85%) of 74 as an oil: NMR δ 0.74 (t, 3 H,J=7), 1.35-1.45 (m, 11 H), 2.00 (t, 2 H, J=7), 2.25 (s, 3 H), 2.28 (s, 3H), 2.55 (s, 6 H), 2.56 (s, 6 H), 2.98-3.08 (m, 8 H), 6.90 (s, 2 H),6.91 (s, 2 H), 7.15-7.44 (m, 15 H). Anal. (C₄₈H₆₁N₃O₄S₂) C, H, N.

EXAMPLE 60

N²-Propylspermidine Trihydrochloride (12). HBr (30% in HOAC, 45 mL), 72(3.70 g, 4.66 mmol), and phenol (8.4 g, 89 mmol) in CH₂Cl₂ (50 mL) werereacted, and product was isolated by the method of 2 to give 1.02 g(74%) of 12 as plates: NMR (D₂O) δ 0.98 (t, 3 H, J=7), 1.66-1.79 (m, 6H), 2.06-2.17 (m, 2 H), 3.01-3.19 (m, 10 H). Anal. (C₁₀H₂₈Cl₃N₃) C, H,N.

EXAMPLE 61

N⁸-Propylspermidine Trihydrochloride (13). HBr (30% in HOAc, 35 mL), 73(2.85 g, 3.59 mmol), and phenol (6.5 g, 69 mmol) in CH₂Cl₂ (30 mL) werereacted, and product was isolated by the method of 2 to give 810 mg(76%) of 13 as plates: NMR (D₂O) δ 0.98 (t, 3 H, J=7), 1.66-1.79 (m, 6H), 2.01-2.12 (m, 2 H), 2.99-3.14 (m, 10 H). Anal. (C₁₀H₂₈Cl₃N₃) C, H,N.

EXAMPLE 62

N¹-Propylhomospermidine Trihydrochloride (18). HBr (30% in HOAc, 20 mL),74 (1.65 g, 2.0 mmol), and phenol (3.6 g, 38 mmol) in CH₂Cl₂ (20 mL)were reacted, and product was isolated by the method of 2 to give 268 mg(43%) of 18 as plates: NMR (D₂O) δ 0.98 (t, 3 H, J=7), 1.66-1.80 (m, 10H), 2.99-3.11 (m, 10 H). HRMS calcd. for C₁₁H₂₈N₃ 202.2283 (free amine,M+H), found. 202.2296 (M+H).

EXAMPLE 63

N¹-Ethylspermidine Trihydrochloride (9). Lithium aluminum hydride (1.6g, 42 mmol) was added to N¹-acetylspermidine dihydrochloride (0.50 g,1.9 mmol) in THF (300 mL) at 0° C., and the mixture was heated at refluxfor 17 hours. The reaction was quenched at 0° C. with H₂O (1.6 mL), 15%NaOH (1.6 mL), and H₂O (4.8 mL). Salts were filtered and washed withTHF, and solvent was removed by rotary evaporation. The residue wasdistilled in a Kugelrohr apparatus under high vacuum (T≦60° C.), and thedistillate was dissolved in EtOH (5 mL) and treated with concentratedHCl (0.5 mL). Recrystallization from aqueous EtOH gave 0.096 g (18%) of9 as crystals: NMR (D₂O) δ 1.30 (t, 3 H, J=7), 1.72-1.83 (m, 4 H),2.05-2.16 (m, 2 H), 3.02-3.19 (m, 10 H). Anal. (C₉H₂₆Cl₃N₃) C, H, N.

EXAMPLE 64

N⁸-Ethylspermidine Trihydrochloride (10). Lithium aluminum hydride (1.73g, 45.6 mmol) and N⁸-acetylspermidine dihydrochloride (0.54 g, 2.1 mmol)in THF (300 mL) were reacted, and product was isolated by the method of9 to furnish 0.164 g (28%) of 10 as crystals: NMR (D₂O) δ 1.29 (t, 3 H,J=7), 1.73-1.83 (m, 4 H), 2.03-2.16 (m, 2 H), 3.05-3.20 (m, 10 H). Anal.(C₉H₂₆Cl₃N₃) C, H, N.

EXAMPLE 65

N¹,N⁴,N⁸,N¹¹-Tetrakis(mesitylenesulfonyl)-N¹,N¹¹-dipropylnorspermine(76). NaH (60%, 3.60 g, 90.0 mmol), 75 [Bergeron et al, J. Med. Chem.,Vol. 37, supra]. (27.5 g, 30.0 mmol), and 1-iodopropane (7.5 mL, 77mmol) in DMF (200 mL) were combined, and the reaction was worked up bythe method of 48. Column chromatography (5:1 toluene/EtOAc) resulted in27.79 g (92%) of 76 as a white foam: NMR δ 0.72 (t, 6 H, J=7), 1.2-1.7(m, 10 H), 2.30 (s, 12 H), 2.55 (s, 24 H), 2.92-3.03 (m, 16 H), 6.93 (s,8 H). Anal. (C₅₁H₇₆N₄O₈S₄) C, H, N.

EXAMPLE 66

N¹,N¹¹-Dipropylnorspermine Tetrahydrochloride (28). HBr (30% in HOAc,500 mL), 76 (27.54 g, 27.5 mmol), and phenol (105 g, 1.12 mol) in CH₂Cl₂(250 mL) were reacted, and product was isolated by the method of 2 togive 7.82 g (68%) of 28 as white plates: NMR (D₂O) δ 0.98 (t, 6 H, J=7),1.64-1.78 (m, 4 H), 2.07-2.21 (m, 6 H), 3.00-3.25 (m, 16 H). Anal.(C₁₅H₄₀Cl₄N₄) C, H, N.

EXAMPLE 67

N-(5-Chloropentyl)-N-ethylmesitylenesulfonamide (77). NaH (80%, 1.34 g,44.7 mmol) was added to 60 [Bergeron et al, J. Med. Chem., Vol. 37,supra]. (7.68 g, 33.8 mmol) in DMF (130 mL) at 0° C. The mixture wasstirred at room temperature for 1 hour, and cooled to 0° C.1,5-Dichloropentane (45 mL, 0.35 mol) was added all at once. Thereaction was stirred at 55° C. for 12 hours and was worked up by themethod of 48. Column chromatography (11.5% EtOAc/hexane) gave 10.54 g(94%) of 77 as an oil: NMR δ 1.07 (t, 3 H, J=8), 1.25-1.37 (m, 2 H),1.47-1.71 (m, 4 H), 2.30 (s, 3 H), 2.60 (s, 6 H), 3.14-3.28 (m, 4 H),3.44 (t, 2 H, J=7), 6.94 (s, 2 H). Anal. (C₁₆H₂₆ClNO₂S) C, H, N.

EXAMPLE 68

3,9,14,20-Tetrakis(mesitylenesulfonyl)-3,9,14,20-tetraazadocosane (79).NaH (80%, 1.14 g, 38.0 mmol) was added to 78 [Bergeron et al, J. Med.Chem., Vol. 37, supra]. (5.77 g, 12.7 mmol) in DMF (75 mL) at 0° C. Themixture was stirred at room temperature for 1 hour, and 77 (10.51 g,31.7 mmol) in DMF (55 mL) was added by cannula. The reaction was stirredat 55° C. for 16 hours and was worked up by the method of 48. Columnchromatography (30% EtOAc/hexane) afforded 12.67 g (96%) of 79 as anoil: NMR δ 0.97-1.12 (m, 10 H), 1.30-1.47 (m, 12 H), 2.29 (s, 12 H),2.56 and 2.58 (2 s, 24 H), 2.97-3.22 (m, 16 H), 6.93 (s, 8 H). Anal.(C₅₄H₈₂N₄O₈S₄) C, H, N.

EXAMPLE 69

3,9,14,20-Tetraazadocosane Tetrahydrochloride (29). HBr (30% in HOAc,195 mL), 79 (12.66 g, 12.1 mmol), and phenol (33.61 g, 0.357 mol) inCH₂Cl₂ (135 mL) were reacted, and product was isolated by the method of2 to provide 4.50 g (81%) of 29 as white crystals: NMR (D₂O) δ 1.28 (t,6 H, J=7), 1.40-1.52 (m, 4 H), 1.66-1.80 (m, 12 H), 3.00-3.14 (m, 16 H).Anal. (C₁₈H₄₆Cl₄N₄).

Analytical Data

2 Anal. calcd. for C₈H₂₄Cl₃N₃: C, 35.77; H, 9.00; N, 15.64. Found: C,35.91; H, 8.96; N, 15.69.

4 Anal. calcd. for C₁₀H₂₈Cl₃N₃: C, 40.48; H, 9.51; N, 14.16. Found: C,40.31; H, 9.36; N, 14.12.

5 Anal. calcd. for C₉H₂₆Cl₃N₃: C, 38.24; H, 9.27; N, 14.87. Found: C,38.15; H, 9.32; N, 14.75.

6 Anal. calcd. for C₁₂H₃₂Cl₃N₃: C, 44.38; H, 9.93; N, 12.94. Found: C,44.42; H, 9.89; N, 12.88.

8 Anal. calcd. for C₉H₂₆Cl₃N₃: C, 38.24; H, 9.27; N, 14.86. Found: C,38.19; H, 9.28; N, 14.79.

9 Anal. calcd. for C₉H₂₆Cl₃N₃: C, 38.24; H, 9.27; N, 14.86. Found: C,38.31; H, 9.23; N, 14.90.

10 Anal. calcd. for C₉H₂₆Cl₃N₃: C, 38.24; H, 9.27 N, 14.86. Found: C,38.28; H, 9.31; N, 14.95.

11 Anal. calcd. for C₁₁H₃₀Cl₃N₃: C, 42.52; H, 9.73; N, 13.52. Found: C,42.59; H, 9.79; N, 13.47.

12 Anal. calcd. for C₁₀H2₈Cl₃N₃: C, 40.48; H, 9.51; N, 14.16. Found C,40.55; H, 9.45; N, 14.18.

13 Anal. calcd. for C₁₀H2₈Cl₃N₃: C, 40.48; H, 9.51; N, 14.16. Found C,40.52; H, 9.52; N, 14.09.

14 Anal. calcd. for C₁₃H₃₄Cl₃N₃: C, 46.09; H, 10.12; N, 12.40. Found: C,46.15; H, 10.17; N, 12.43.

15 Anal. calcd. for C₈H₂₄Cl₃N₃: C, 35.77; H, 9.00; N, 15.64; S, 39.59.Found: C, 35.88; H, 8.91; N, 15.68; S, 39.48.

16 Anal. calcd. for C₁₀H₂₈Cl₃N₃: C, 40.48; H, 9.51; N, 14.16. Found: C,40.45; H, 9.44; N, 14.09.

17 Anal. calcd. for C₁₂H₃₂Cl₃N₃: C, 44.38; H, 9.93; N, 12.94. Found: C,44.49; H, 9.98; N, 12.96.

19 Anal. calcd. for C₁₄H₃₆Cl₃N₃: C, 47.66; H, 10.29; N, 11.91. Found: C,47.70; H, 10.21; N, 11.86.

20 Anal. calcd. for C₉H₂₆Cl₃N₃: C, 38.24; H, 9.27; N, 14.87. Found: C,38.28; H, 9.22; N, 14.82.

21 Anal. calcd. for C₁₁H₃₀Cl₃N₃: C, 42.52; H, 9.73; N, 13.52. Found: C,42.52; H, 9.69; N, 13.58.

22 Anal. calcd. for C₁₃H₃₄Cl₃N₃: C, 46.09; H, 10.12; N, 12.40. Found: C,46.20; H, 10.08; N. 12.47.

23 Anal. calcd. for C₁₅H₃₈Cl₃N₃: C, 49.11; H, 10.44; N, 11.45. Found: C,49.02; H, 10.40; N, 11.42.

24 Anal. calcd. for C₁₀H₂₈Cl₃N₃: C, 40.48; H, 9.51; N, 14.16; Cl, 35.85.Found: C, 40.63; H, 9.44; N, 14.16; Cl, 35.70.

25 Anal. calcd. for C₁₂H₃₂Cl₃N₃: C, 44.38; H, 9.93; N, 12.94. Found: C,44.33; H, 9.90; N, 12.89.

26 Anal. calcd. for C₁₄H₃₆Cl₃N₃: C, 47.66; H, 10.28; N, 11.91; Cl,30.15. Found: C, 47.69; H, 10.24; N, 11.96; Cl, 30.08.

27 Anal. calcd. for C₁₆H₄₀Cl₃N₃: C, 50.46; H, 10.59; N, 11.03. Found: C,50.49; H, 10.55; N, 11.07.

28 Anal. calcd. for C₁₅H₄₀Cl₄N₄: C, 43.07; H, 9.64; N, 13.39. Found: C,43.24; H, 9.57; N, 13.44.

29 Anal. calcd. for C₁₈H₄₆Cl₄N₄: C, 46.96; H, 10.07; N, 12.17. Found: C,47.08; H, 9.98; N, 12.18.

30 Anal. calcd. for C₃₃H₄₇N₃O₆S₃: C, 58.47; H, 6.99; N, 6.20. Found: C,58.36; H, 6.95; N, 6.18.

31 Anal. calcd. for C₄H₄₉N₃O₆S₃: C, 59.02; H, 7.14; N, 6.07. Found: C,58.74; H, 7.12; N, 5.99.

32 Anal. calcd. for C₃₅H₅₁N₃O₆S₃: C, 59.55; H, 7.28; N, 5.95. Found: C,59.34; H, 7.29; N, 5.92.

33 Anal. calcd. for C₃₆H₅₃N₃O₆S₃: C, 60.05; H, 7.42; N, 5.84. Found: C,59.88; H, 7.41; N, 5.80.

36 Anal. calcd. for C₁₃H₁₈N₂O₂S: C, 58.62; H, 6.81; N, 10.52. Found: C,58.52; H, 6.86; N, 10.46.

38 Anal. calcd. for C₁₈H₂₅N₃O₂S: C, 62.22; H, 7.25; N, 12.09; S, 8.87.Found: C, 62.24; H, 7.28; N, 11.99.

39 Anal. calcd. for C₁₉H₂₇N₃O₂S: C, 63.13; H, 7.53; N, 11.62; S, 8.87.Found: C, 63.31; H, 7.68; N, 11.43; S, 8.97.

43 Anal. calcd. for C₃₅H₅₁N₃O₆S₃: C, 59.55; H, 7.28; N, 5.95. Found: C,59.50; H, 7.33; N, 5.88.

44 Anal calcd. for C₃₇H₅₅N₃O₆S₃: C, 60.54; H, 7.55; N, 5.72. Found: C,60.64; H, 7.54; N, 5.73.

45 Anal. calcd. for C₃₆H₅₃N₃O₆S₃: C, 60.05; H, 7.42; N, 5.84. Found: C,59.79; H, 7.32; N, 5.70.

46 Anal. calcd. for C₃₉H₅₉N₃O₆S₃: C, 61.55; H, 7.68; N, 5.52. Found: C,61.52; H, 7.79; N, 5.55.

48 Anal. calcd. for C₃₈H₅₇N₃O₆S₃: C, 61.01; H, 7.68; N, 5.62. Found: C,61.22; H, 7.76; N, 5.56.

49 Anal. calcd. for C₄₀H₆₁N₃O₆S₃: C, 61.90; H, 7.92; N, 5.41. Found: C,61.71; H, 7.86; N, 5.35.

50 Anal. calcd. for C₃₇H₅₅N₃O₆S₃: C, 60.54; H, 7.55; N, 5.72. Found: C,60.26; H, 7.61; N, 5.63.

51 Anal. calcd. for C₃₉H₅₉N₃O₆S₃: C, 61.47; H, 7.80; N, 5.51. Found: C,61.27; H, 7.89; N, 5.44.

52 Anal. calcd. for C₄₁H₆₃N₃O₆S₃: C, 62.32; H, 8.04; N, 5.32. Found: C,62.19; H, 8.00; N, 5.33.

54 Anal. calcd. for C₄₂H₆₅N₃O₆S₃H₂O: C, 61.35; H, 8.21; N, 5.11. Found:C, 61.34; H, 8.07; N, 5.05.

55 Anal. calcd. for C₃₉H₅₉N₃O₆S₃: C, 61.47; H, 7.80; N, 5.51. Found: C,61.54; H, 7.79; N, 5.51.

56 Anal. calcd. for C₄₁H₆₃N₃O₆S₃: C, 62.32; H, 8.04; N, 5.32; S, 12.17.Found: C, 62.40; H, 8.08; N, 5.25; S, 12.07.

57 Anal. calcd. for C₄₃H₆₇N₃O₆S₃: C, 63.12; H, 8.25; N, 5.14. Found: C,63.21; H, 8.23; N, 5.04.

63 Anal. calcd. for C₂₄H₃₆N₂O₄S₂: C, 59.97; H, 7.55; N, 5.83. Found: C,59.83; H, 7.56; N, 5.76.

64 Anal. calcd. for C₄₀H₆₁N₃O₆S₃: C, 61.90; H, 7.92; N, 5.41. Found: C,62.03; H, 7.97; N, 5.33.

65 Anal. calcd. for C₁₂H₁₉NO₂S: C, 59.72; H, 7.93; N, 5.80. Found: C,59.69; H, 7.88; N, 5.80.

66 Anal. calcd. for C₁₅H₂₄BrNO₂S: C, 49.72; H, 6.68; N, 3.87. Found C,49.97; H, 6.76; N, 3.83.

67 Anal. calcd. for C16H₂₆BrNO₂S: C, 51.06; H, 6.96; N, 3.72. Found: C,51.17; H, 6.95; N, 3.74.

68 Anal. calcd. for C₂₂H₂₄N₂: C, 83.50; H, 7.64; N, 8.85. Found C,83.42; H, 7.67; N, 8.86.

70 Anal. calcd. for C₃₁H₃₄N₂O₂S: C, 74.67; H, 6.87; N, 5.62. Found: C,74.62; H, 6.89; N, 5.54.

71 Anal. calcd. for C₃₂H₃₆N₂O₂S: C, 74.97; H, 7.08; N, 5.46. Found: C,74.71; H, 7.12; N, 5.51.

72 Anal. calcd. for C₄₇H₅₉N₃O₄S₂: C, 71.09; H, 7.49; N, 5.29. Found C,71.35; H, 7.53; N, 5.18.

73 Anal. calcd. for C₄₇H₅₉N₃O₄S₂: C, 71.09; H, 7.49; N, 5.29. Found C,71.16; H, 7.46; N, 5.33.

74 Anal. calcd. for C₄₈H₆₁N₃O₄S₂: C, 71.34; H, 7.61; N, 5.20. Found C,71.22; H, 7.64; N, 5.10.

76 Anal. calcd. for C₅₁H₇₆N₄O₈S₄: C, 61.17; H, 7.65; N, 5.59. Found: C,61.21; H, 7.67; N, 5.58.

77 Anal. calcd. for C₁₆H₂₆ClNO₂S: C, 57.90; H, 7.90; N, 4.22. Found: C,57.98; H, 7.82; N, 4.27.

79 Anal. calcd. for C₅₄H₈₂N₄O₈S₄: C, 62.16; H, 7.92; N, 5.37. Found: C,62.30; H, 7.86; N, 5.37.

What is claimed is:
 1. A polyamine which does not occur in nature havingthe formula:

or a salt thereof with a pharmaceutically acceptable acid wherein: R₁-R₅may be the same or different and are alkyl, aryl, aryl alkyl,cyclo-alkyl or hydrogen; at least one of said R₁, and R₂ and at leastone of said R₄ and R₅ are not hydrogen, and any of said alkyl chains mayoptionally be interrupted by at least one etheric oxygen atom, excludingN¹,N³-diethylspermidine and N¹,N³-dipropylspermidine; N¹,N² and N³ arenitrogen atoms capable of protonation at physiological pH's; and A and Bmay be the same or different and are bridging groups selected from thegroup consisting of alkylene branched alkylene, cycloalkylene,arlalkylene or unsubstituted heterocyclic bridging groups whicheffectively maintain the distance between the nitrogen atoms such thatthe polyamine: (i) is capable of uptake by a target cell uponadministration of the polyamine to a human or non-human animal; and (ii)upon uptake by said target cell, competitively binds via anelectrostatic interaction between the positively charged nitrogen atomsto substantially the same biological counter-anions as the intracellularnatural polyamines in the target cell, provided that where A or B is aheterocyclic bridging group, the bridging group is an unsubstitutedheterocyclic group incorporating said N¹,N² or N³ atom in theheterocyclic ring as an unsubstituted N atom; said polyamine, uponbinding to the biological counter-anion in the cell, functions in amanner biologically different than said intracellular polyamines.
 2. Apolyamine according to claim 1, upon binding to said biologicalcounter-anion in said cell, exerting an anti-neoplastic function.
 3. Thepolyamine according to claim 1 having the formula:R₁—N¹H—(CH₂)₃—N²H—(CH₂)₃—N³H—R₂ wherein: R₁ and R₂ may be the same ordifferent and are alkyl having up to 10 carbon atoms.
 4. The polyamineof claim 3 wherein R₁=R₂=methyl.
 5. The polyamine of claim 3 whereinR₁=R₂=ethyl.
 6. The polyamine of claim 3 wherein R₁=R₂=n-propyl.
 7. Thepolyamine of claim 3 wherein R₁=H and R₂=ethyl.
 8. The polyamine ofclaim 3 wherein R₁=H and R₂=n-propyl.
 9. The polyamine according toclaim 1 having the formula: R₁—N¹H—(CH₂)₃—N²H—(CH₂)₄—N³H—R₂ wherein: R₁and R₂ may be the same or different and are alkyl having up to 10 carbonatoms.
 10. The polyamine of claim 9 wherein R₁=R₂=methyl.
 11. Thepolyamine of claim 9 wherein R₁=R₂=ethyl.
 12. The polyamine of claim 9wherein R₁=R₂=n-propyl.
 13. The polyamine of claim 9 wherein R₁=ethyland R₂=H.
 14. The polyamine of claim 9 wherein R₁=H and R₂=ethyl. 15.The polyamine of claim 9 wherein R₁=n-propyl and R₂=H.
 16. The polyamineof claim wherein R₁=H and R₂=n-propyl.
 17. The polyamine according toclaim 1 having the formula: R₁—N¹H—(CH₂)₄—N²H—(CH₂)₄—N³H—R₂ wherein: R₁and R₂ may be the same or different and are alkyl having up to 10 carbonatoms.
 18. The polyamine of claim 17 wherein R₁=R₂=methyl.
 19. Thepolyamine of claim 17 wherein R₁=R₂=ethyl.
 20. The polyamine of claim 17wherein R₁=R₂=n-propyl.
 21. The polyamine of claim 17 wherein R₁=H andR₂=ethyl.
 22. The polyamine of claim 17 wherein R₁=H and R₂=n-propyl.23. The polyamine according to claim 1 having the formula:R₁—N¹H—(CH₂)₄—N²H—(CH₂)₅—N³H—R₂ wherein: R₁ and R₂ may be the same ordifferent and are alkyl having up to 10 carbon atoms.
 24. The polyamineof claim 23 wherein R₁=R₂=methyl.
 25. The polyamine of claim 23 whereinR₁=R₂=ethyl.
 26. The polyamine of claim 23 wherein R₁=R₂=n-propyl. 27.The polyamine according to claim 1 having the formula:R₁—N¹H—(CH₂)₅—N²H—(CH₂)₅—N³H—R₂ wherein: R₁ and R₂ may be the same ordifferent and are alkyl having up to 10 carbon atoms.
 28. The polyamineof claim 23 wherein R₁=R₂=methyl.
 29. The polyamine of claim 23 whereinR₁=R₂=ethyl.
 30. The polyamine of claim 23 wherein R₁=R₂=n-propyl.
 31. Apharmaceutical composition in unit dosage form comprising apharmaceutically acceptable carrier and a pharmaceutically effectiveamount of a polyamine of claim 1 or a salt thereof with apharmaceutically acceptable acid.
 32. The pharmaceutical composition ofclaim 31 comprising an amount of said polyamine or salt pharmaceuticallyeffective to treat a human or non-human patient afflicted with tumorcells sensitive to said polyamine or salt thereof.
 33. A method oftreating a human or non-human patient in need thereof comprisingadministering thereto a pharmaceutically effective amount of a polyamineof claim 1 or a salt thereof with a pharmaceutically acceptable acid.34. The method according to claim 33 comprising administering to saidpatient afflicted with tumor cells sensitive to said polyamine or saltthereof an amount of said polyamine or salt thereof pharmaceuticallyeffective to inhibit the growth of said tumor cells.
 35. A polyamineaccording to claim 1, wherein at least one of A and B is anunsubstituted heterocyclic bridging group.